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CyAn™ ADP User Guide - Weatherall Institute of Molecular Medicine

1. Pi summit 4 1 Licensed to DakoCytomation User jamess Database DB1 sum File Edit View Histogram Gate Workspace Tools Help 7 Protocol 1 7 Workspace 1 _ x Protocol 1 Sample 3 c Desktop ADP P3_02_JUL_2004 35 cells tube 2 fcs oi aie A Sample Histogram Gating Workspace H IS ile Template ra J sample 3 EI E Sample properties Sample 3 A Item value z Q Sample Number 35 A Q Samp le Sample 3 a Q source cells amp Q Date 02 Jul 200 aisha E Q Total Events 23506 0 64 128 192 256 Q Elapsed time 00h 01m 57 FS Lin Average Rate 200 91 eps Ce TE a Region Count zml all Region count Hist aal eege Total 23234 100 00 100 00 Total 19986 100 00 100 00 Q Parameter 2 Time MSW 5 Al s Q Parameter 3 Pulse Widt Q Parameter 4 FS Lin e Database samples 7 EI Q Parameter 5 3S Lin HI Q Parameter 6 FITC Log Name Path Q Parameter Fees PE Log s analysis Q Parameter 8 PE Texas H C Sampie 1 C Docum Q Parameter 9 PE Cy5 Log H E amie 2 C Docum Q Parameter 1 PE Cy Log H 3 C Docum Q Parameter 1 Violet 1 4 i Sample compensation Parameter FITC Log Region Count zml sall Total 23506 100 00 100 00 4 4 F E Ready Ma Offline Protocol 1 smpe OE 0 00 Compensation Summit Software performs full 9 x 9 inter laser compensation of fluorescence parameters Data saved as a sample file in Summit will reta
2. CyAn ADP User Guide iii Contacting DakoCytomation DakoCytomation should be contacted immediately for assistance in the event of any instrument malfunction For further information please contact your local DakoCytomation office Australia Tel 2 9316 4633 Fax 2 9316 4773 Austria Tel 0800 0800 7153 Fax 0800 0800 7154 Belgium Tel 016 38 72 20 Fax 016 38 72 21 Canada Tel 905 858 8510 Fax 905 858 8801 Czech Republic Tel 420 541 423 710 Fax 420 541 423 711 Denmark Head Office Tel 44 85 95 00 Fax 44 85 95 95 Sales Tel 44 85 97 56 Fax 44 85 84 29 France Tel 1 30 50 00 50 Fax 1 30 50 00 11 Germany Tel 040 69 69 470 Fax 040 69 52 741 Italy Tel 02 58 078 1 Fax 02 58 078 292 Japan Tel 075 211 3655 Fax 075 211 1755 The Netherlands Tel 020 42 11 100 Fax 020 42 11 101 Norway Tel 23 14 05 40 Fax 23 14 05 42 Poland Tel 058 661 1879 Fax 058 661 3390 Spain Tel 93 499 05 06 Fax 93 499 02 08 Sweden Tel 08 556 20 600 Fax 08 556 20 619 Switzerland Tel 041 760 11 66 Fax 041 760 11 77 United Kingdom Tel 0 1 353 66 99 11 Fax 0 1 353 66 89 89 Technical Support Tel 0 1 353 66 99 65 United States of America Carpinteria California Tel 805 566 6655 Fax 805 566 6688 Technical Support Tel 800 424 0021 Customer Service Tel 800 235 5763 United States of America Fort Collins Colorado Flow Instrumentation Tel 800 822 9902 Fax 970 226 0107 CyAn ADP
3. Relative Humidity 20 to 80 RH non condensing CyAn ADP UV Model Coherent Enterprise Laser Power Requirements A dedicated 208 240V single phase 50 amp circuit is required The laser is hardwired into a junction box with fused disconnect The junction box must be within 6 feet of the CyAn ADP An electrician must be available to connect and ensure adequate power for the Enterprise laser on the first day of CyAn ADP installation CyAn ADP User Guide CyAn ADP UV Model Coherent Enterprise Laser Ambient Air and Cooling Water Specifications The Enterprise laser is a water cooled laser that requires very stable temperatures of the ambient air and the cooling water CyAn ADP UV Model Heat Exchanger The CyAn ADP UV model comes with a Coherent Laser Pure 5 LP5 water to air heat exchanger The LP5 generates 17 000 BTU hour that needs to be dissipated as efficiently as possible Laser stability is dependent on the constant temperature of the water circulating through the laser cavity Because the water is cooled through a water to air heat exchanger the ambient temperature and humidity must be constant Heating and air conditioning vents or fans are not recommended directly above the CyAn ADP UV model because of the resulting temperature fluctuation Table 2 3 lists some helpful guidelines for optimum laser performance Table 2 3 Coherent LP5 Optimum Conditions Item Description Ambient 2 C Temperature
4. 10 10 10 10 APC CyT Comp 5324 1000 S T 5324 10000 1 64 2 0 om op o on o om K 0 Qo 2606 67 73 J624 6807 1718_3227 13824 1 00 1700 _31 93 86 CyAn ADP User Guide Dakocytomation Running the All Stains Sample Finally run the all stains or experimental sample This sample can be analyzed using dot plots containing any combination of the 5 parameters FITC PE PE Cy5 APC and APC Cy7 If compensation has been properly set the populations will be aligned so that the positive and negative populations can be easily identified Again when running the all stains sample verify that the quad regions are aligned along the first log decade marks L G1 R1 All Stains G1 R1 All Stains CyS Comp PE D E coun 5 5853 10000 53 23 237 14 100 00 1 00 237 14 1616 2742 1 11 2067 4052 6876 77 74 245 92 25 30 1 72 340 D D 0 00 0 00 102 107 APC C Comp 8461 1 00 237 14 662 11 57 177 83 24 AJ 378 237 3 40 2 Q03 11 14 2 64 101 102 10 FITC Come Total 3 10000 170 10000 5829 36 52 10000 5829 1 00 R20 62 105 100 18434 24 34 1 04 27 38 4 55 1 60 24 58 R21 8 1 10 14 33 13824 GREG 77 74 67 32 2965 72 34 115 48 R22 om 5515 mm 1 00 77 Ga 1 78 2 55 2654 1 24 1 00 R23 1956 3319 11140 1 00 6 og 5624 5 23 3317 9660 1 00 Putting Compensation Into Practice This tutorial has demonstrated how to perform a 5 color experiment involving compensati
5. Fluorescent Light Signal Electrical Output 0 10 Volts KE Range Infinite 00 values DAAT Analog to Digital Conversion Digital Output Channels Range Finite of Values e g 256 or 1024 The Digital Signal and Software Data Display The number of possible channels to which a particle s signal can be assigned is a finite amount a discrete as opposed to a continuous distribution and is dependent upon the resolution of the ADC For example a 10 bit ADC can bin the electronic signal or voltage into one of 1024 possible channels or 2 n where n 10 A 12 bit ADC has a range up to 4096 channels or 2 10 This range of available channels determines the visual resolution of the data when analyzed within a histogram or dot plot Increased Voltage Recorded Higher Channel Assignment Converted Analog Signals 1 Volt 5 Volts 9 Volts 0 1 o 64 128 192 256 Digital Scale Based on the analog signal recorded from a particle or cell the digital output will be assigned to a particular channel Resolution of the data or the potential to distinguish between two events is partially dependent upon the number of channels available For example if only a single channel were available events within a histogram would be digitally or visually indistinguishable since they would all fall within the same channel For a gaussian or a symmetrically distributed population a two channel situation would show half the events
6. FSC Area versus Pulse Width The FSC Area vs PW method provides doublet information on both axes of the dual parameter histogram To set it up you simply select Area on the FSC analog to digital converter ADC In Summit create a dual parameter histogram plotting FSC area vs Pulse Width Define a gated region on the main singlet population and include this region in your Boolean logic sort decision FSC Area versus SSC Area The FSC Area vs SSC Area also provides doublet information on both axes To set it up make sure both FSC and SSC ADC s are set to Area Create a FSC area vs SSC area histogram in Summit and gate on the main singlet population CyAn ADP User Guide 107 Doublet Discrimination FSC Int vs Pulse W idth e Set FSC ADC to Integrate e Create FSC vs PW histogram in Summit e Gate onthe singlet population for sorting FSC Int vs SSCdnt e Set switches on FSC and SC ADC s to Integrate Create FSC vs SSC histogram in Summit Gate on the singlet population for sorting Histogram Scaling A number of manual and auto scaling features are available for histograms in Summit The following tutorial explains the various scaling options and how each functions to enhance data display and analysis In the Histogram Properties dialog the Scale list item provides access to all the manual and auto scale options 108 CyAn ADP User Guide vakocytomation Histogram Properties General Manual
7. CYAN zl O New Size 149504 bytes Last modified 06 04 2004 Read only No Ee X Cancel V Show this dialog at startup CyAn ADP User Guide 21 4 Click the Instrument tab to view the CyAn Control Panel xf V On 488nm Opened V On 405mm m Opened V On 635nm m Opened Shutdown e Backtlush Vacuum ge Debubble Fluidics of Cleanvrinse Sheath Management System A Laser LG amp Interlock Press Vac oh S e ae qe o e S a e Sheath Clean Waste Cl SIE a m Preset if 5 9 5 Check the status of the sheath container and the waste container by viewing the Sheath and Waste levels in the CyAn Control Panel Make sure the sheath container is at least half full and the waste container is at least half empty If necessary fill the sheath container and empty the waste container as described in Section 5 Fluids Management WARNING Remove the waste container and the sheath container from the cart before emptying or filling Dispose of the contents of the waste container in accordance with local state and federal regulations WARNING Do not drop the waste or sheath container on the Sheath Management System Doing so may result in improper calibration of the load cells 6 Click Startup on the Cyan Control Panel 22 CyAn ADP User Guide Ge DakoCytomation 7 On the CyAn Control Panel make sure the desired lasers are
8. Green comp Green 0 3 Yellow comp 0 3 Blue comp Blue comp Blue 0 0 Yellow comp 0 3 Green comp However that introduces an interesting recursion because compensated Yellow is dependent on compensated Green which is dependent on compensated Yellow However if you disallow the recursion you can substitute out the equations and get the following without performing negative compensation Yellow comp Yellow 0 3 Green 0 3 Yellow 0 3 Blue 0 0 Blue 0 0 Yellow 0 3 Green Green comp Green Yellow 0 3 Green Blue comp Blue 0 0 0 3 Blue 0 3 Yellow 0 3 Green 0 0 Blue 0 3 Blue 0 0 Yellow 0 3 Green 0 0 Blue 0 3 Green 0 3 Yellow Performing the calculation for compensated Yellow on the same events for this example would yield the following results CyAn ADP User Guide 51 1 Yellow comp 0 0 3 0 0 3 0 0 3 0 0 0 0 0 0 0 0 3 0 0 0 2 Yellow comp 10 0 3 3 0 3 10 0 3 0 0 0 0 0 0 10 0 3 3 10 3 Yellow comp 3 0 3 10 0 3 3 0 3 3 0 0 3 0 0 3 0 3 10 0 54 4 Yellow comp 0 0 3 3 0 3 0 0 3 10 0 0 10 0 0 0 0 3 3 0 0 5 Yellow comp 10 0 3 6 0 3 10 0 3 10 0 0 1
9. PMTs using various combinations of filters for example bandpass BP shortpass SP or longpass LP and dichroic mirrors The filters are designed and configured to provide the best optical resolution of the signals elicited from each cell or particle Because the filters are configured to allow only certain wavelengths of light to reach a given detector each PMT becomes specific for detecting fluorescent compounds with compatible emission spectra such as FITC PE PE Cy5 GFP or Texas Red signal gt i il PMT PMT PMT Dichroic filter BP SP or LP filter With the aid of an amplifier PMTs convert this reflected or fluorescent light into an electrical signal voltage which describes the intensity of light from each cell Signals are usually quantified and reported within the range of 0 10 volts Keep in mind the number of possible values within this range is infinite For example cells can elicit signals with values of 2 3 2 456 4 56897 volts etc Analog values recorded along this continuous distribution are later assigned to a 98 CyAn ADP User Guide Gi ocytomation specific channel number or bin with the aid of an Analog to Digital Converter ADC The end result is a digital description of a single event that contains an intensity value for each fluorescent or light scatter color The data s digital signal can then be visualized and analyzed using a number of methods such as a histogram or dot plot
10. SHEATH MANAGEMENT SYSTEM RESET SHEATH LEVEL CLEANER LEVEL WASTE LEVEL Bei ww oy e UI tow empty O DakoCytomation When the waste fluid level is high the green OK LED changes to a flashing amber LED beside the HIGH label SHEATH MANAGEMENT SYSTEM RESET SHEATH LEVEL CLEANER LEVEL DG e ok wo WASTE LEVEL A LOW P LOW m EMPTY EMPTY TO DakoCytomation CyAn ADP User Guide 29 When the sheath or cleaner level changes from low to empty the flashing amber LED changes to a red LED beside the EMPTY label SHEATH MANAGEMENT SYSTEM CLEANER LEVEL WASTE LEVEL wor RESET SHEATH LEVEL E3 ww oy LI tow e empty J LOW TO DakoCytomation When the waste fluid level changes from high to full the flashing amber LED changes to a red LED beside the FULL label SHEATH MANAGEMENT SYSTEM RESET CLEANER LEVEL WASTE LEVEL TO DakoCytomation 30 CyAn ADP User Guide Mos oCytomation Table 5 1 lists the indicators in the CyAn Control Panel user interface and the message text for each error or status condition Table 5 1 CyAn ADP Control Panel Indicators CyAn Control Panel Message Text e A Laser Message 101 The 621 Laser has reported a fault Please check the cooling lines and interlocks on the 621 Laser Message 102 Your CyAn cover is open The laser shutters have been closed and fluidic system is shutdown Please close your cover and restart Message 301 C
11. 1999 Promoter elements of vav drive transgene expression in vivo throughout the hematopoietic compartment Blood 94 1855 Okuno Y H lwasaki C S Huettner H S Radomska D A Gonzalez D G Tenen and K Akashi 2002 Differential regulation of the human and murine CD34 genes in hematopoietic stem cells PNAS 99 6246 Okuno Y C S Huettner H S Radomska V Petkova H Iwasaki K Akashi and D G Tenen 2002 Distal elements are critical for human CD34 expression in vivo Blood 100 4420 Pyatt D W W S Stillman Y Yang S Gross J H Zheng and R D Irons 1999 An essential role for NF xB in human CD34 bone marrow cell survival Blood 93 3302 Radomska H S D A Gonzalez Y Okuno H Iwasaki A Nagy K Akashi D G Tenen and C S Huettner 2002 Transgenic targeting with regulatory elements of the human CD34 gene Blood 100 4410 Reddy G P C Y Tiarks L Pang J Wuu C C Hsieh and P J Quesenberry 1997 Cell cycle analysis and synchronization of pluripotent hematopoietic progenitor stem cells Blood 90 2293 Santulli Marotto S M W Retter R Gee M J Mamula and S H Clarke 1998 Autoreactive B cell regulation peripheral induction of developmental arrest by lupus associated autoantigens Immunity 8 209 Schaniel C L Bruno F Melchers and A G Rolink 2002 Multiple hematopoietic cell lineages develop in vivo from transplanted Pax5 deficient pre B l cell clones Blood 99 472 S
12. A Goodell and K K Hirschi 2003 Distinct progenitor populations in skeletal muscle are bone marrow derived and exhibit different cell fates during vascular regeneration J Clin Invest 111 71 Manz M G T Miyamoto K Akashi and I L Weissman 2002 Prospective isolation of human clonogenic common myeloid progenitors PNAS 99 11872 Martin F and J F Kearney 2000 Positive selection from newly formed to marginal zone B cells depends on the rate of clonal production CD19 and btk Immunity 12 39 CyAn ADP User Guide 141 McKinney Freeman S L K A Jackson F D Camargo G Ferrari F Mavilio and M A Goodell 2002 Muscle derived hematopoietic stem cells are hematopoietic in origin PNAS 99 1341 Metcalf D L Di Rago and S Mifsud 2002 Synergistic and inhibitory interactions in the in vitro control of murine megakaryocyte colony formation Stem Cells 20 552 Mikkola H K A Y Fujiwara T M Schlaeger D Traver and S H Orkin 2003 Expression of CD41 marks the initiation of definitive hematopoiesis in the mouse embryo Blood 101 508 Moore K A H Ema and R Lemischka 1997 In vitro maintenance of highly purified transplantable hematopoietic stem cells Blood 89 4337 Nilsson S K M S Dooner and P J Quesenberry 1997 Synchronized cell cycle induction of engrafting long term repopulating stem cells Blood 90 4646 Ogilvy S D Metcalf L Gibson M L Bath A W Harris and J M Adams
13. Data can be binned and displayed using 256 512 1024 2048 or 4096 channels These settings are available in the Histogram Properties dialog Histogram Properties General Resolution 256 v Smoothing Axis Axis Ticks Univariates Scale Bin Resolution A Close CyAn ADP User Guide 101 As the channel resolution for a histogram is increased the data becomes dispersed within more channels along the axis and the Y axis is rescaled to reflect the reduced event count per channel This can result in the data profile appearing more jagged Ei Resolution Example gt EE Resolution Example 1024 2048 3072 4096 FSC Total 50000 100 00 100 00 When the resolution is changed auto scaling is performed which maintains the overall data profile Although useful this can be visually misleading unless the counts along the Y axis are taken into consideration E Resolution Example pa 78 0 1024 2048 3072 4096 FSC Region Count hist za Total 50000 100 00 100 00 gt lt 102 CyAn ADP User Guide Moz oCytomation As the resolution on the same histogram is changed using a fixed count scale the data will flatten along the X axis This is expected since the same number of events is now distributed within an increased number of channels in the histogram increased number of channels for better resolution of the data set but with fewer events per channel Ultimately
14. G P Rueda I Casal and C Leclerc 2002 CD8a CD11b dendritic cells present exogenous virus like particles to CD8 T cells and subsequently express CD8a and CD205 molecules J Exp Med 195 1233 Nishizawa K C Freund J Li G Wagner and E L Reinherz 1998 Identification of a proline binding motif regulating CD2 triggered T lymphocyte activation PNAS 95 14897 Ohdan H Y G Yang A Shimizu K G Swenson and M Sykes 1999 Mixed chimerism induced without lethal conditioning prevents T cell and anti Gal a 1 3Gal mediated graft rejection J Clin Invest 104 287 Ouyang W M Lohning Z Gao M Assenmacher S Ranganath A Radbruch and K M Murphy 2000 Stat6 independent GATA 3 autoactivation directs IL 4 independent Th2 development and commitment Immunity 12 27 Pestano G A Y Zhou L A Trimble J Daley G F Weber and H Cantor 1999 Inactivation of misselected CD8 T cells by CD8 gene methylation and cell death Science 284 1187 Poussier P T Ning D Banerjee and M Julius 2002 A unique subset of self specific intraintestinal T cells maintains gut integrity J Exp Med 195 1491 Rengarajan J K A Mowen K D McBride E D Smith H Singh and L H Glimcher 2002 Interferon regulatory factor 4 IRF4 interacts with NFATc2 to modulate interleukin 4 gene expression J Exp Med 195 1003 144 CyAn ADP User Guide Gi oCytomation Reuther Madrid J Y D Kashatus S Chen X Li J Westwick R
15. H von Boehmer and A C Hayday 2001 The biological activity of natural and mutant pTa alleles J Exp Med 194 695 Girardi M D E Oppenheim C R Steele J M Lewis E Glusac R Filler P Hobby B Sutton R E Tigelaar and A C Hayday 2001 Regulation of cutaneous malignancy by 6 T cells Science 294 605 Gnjatic S Y Nagata E Jager E Stockert S Shankara B L Roberts G P Mazzara S Y Lee P R Dunbar B Dupont V Cerundolo G Ritter Y T Chen A Knuth and L J Old 2000 Strategy for monitoring T cell responses to NY ESO 1 in patients with any HLA class allele PNAS 97 10917 Goldrath A W P V Sivakumar M Glaccum M K Kennedy M J Bevan C Benoist D Mathis and E A Butz 2002 Cytokine requirements for acute and basal homeostatic proliferation of naive and memory CD8 T cells J Exp Med 195 1515 Grogan J L M Mohrs B Harmon D A Lacy J W Sedat and R M Locksley 2001 Early transcription and silencing CyAn ADP User Guide 143 of cytokine genes underlie polarization of T helper cell subsets Immunity 14 205 Hildeman D A T Mitchell T K Teague P Henson B J Day J Kappler and P C Marrack 1999 Reactive oxygen species regulate activation induced T cell apoptosis Immunity 10 735 Hirst C E M S Buzza C H Bird H S Warren P U Cameron M Zhang P G Ashton Rickardt and P Bird 2003 The intracellular granzyme B inhibitor proteinase inh
16. J Davis H S Earp C Y Wang and A S Baldwin Jr 2002 The p65 RelA subunit of NF B suppresses the sustained antiapoptotic activity of Jun kinase induced by tumor necrosis factor Mol Cell Biol 22 8175 Riese R J G P Shi J Villadangos D Stetson C Driessen A M Lennon Dumenil C L Chu Y Naumov S M Behar H Ploegh R Locksley and H A Chapman 2001 Regulation of CD1 function and NK1 1 T cell selection and maturation by cathepsin S Immunity 15 909 Seo S J M L Fields J L Buckler A J Reed L Mandik Nayak S A Nish R J Noelle L A Turka F D Finkelman A J Caton and J Erikson 2002 The impact of T helper and T regulatory cells on the regulation of anti double stranded DNA B cells Immunity 16 535 Shires J E Theodoridis and A C Hayday 2001 Biological insights into TCRy6 and TCRaB intraepithelial lymphocytes provided by serial analysis of gene expression SAGE mmunity 15 419 Strong J Q Wang and N Killeen 2001 Impaired survival of T helper cells in the absence of CD4 PNAS 98 2566 Szmania S A Galloway M Bruorton P Musk G Aubert A Arthur H Pyle N Hensel N Ta L Lamb Jr T Dodi A Madrigal J Barrett J Henslee Downey and F van Rhee 2001 Isolation and expansion of cytomegalovirus specific cytotoxic T lymphocytes to clinical scale from a single blood draw using dendritic cells and HLA tetramers Blood 98 505 Teague T K D Hildeman R
17. M Kedl T Mitchell W Rees B C Schaefer J Bender J Kappler and P Marrack 1999 Activation changes the spectrum but not the diversity of genes expressed by T cells PNAS 96 12691 Teague T K B C Schaefer D Hildeman J Bender T Mitchell J W Kappler and P Marrack 2000 Activation induced inhibition of interleukin 6 mediated T cell survival and signal transducer and activator of transcription 1 signaling J Exp Med 191 915 Tibaldi E V R Salgia and E L Reinherz 2002 CD2 molecules redistribute to the uropod during T cell scanning Implications for cellular activation and immune surveillance PNAS 99 7582 Usherwood E J R J Hogan G Crowther S L Surman T L Hogg J D Altman and D L Woodland 1999 Functionally heterogeneous CD8 T cell memory is induced by Sendai virus infection of mice J Virol 73 7278 Vivien L C Benoist and D Mathis 2001 T lymphocytes need IL 7 but not IL 4 or IL 6 to survive in vivo Int Immunol 13 763 Voehringer D M Koschella and H Pircher 2002 Lack of proliferative capacity of human effector and memory T cells expressing killer cell lectinlike receptor G1 KLRG1 Blood 100 3698 Wagner D H Jr G Vaitaitis R Sanderson M Poulin C Dobbs and K Haskins 2002 Expression of CD40 identifies a unique pathogenic T cell population in type 1 diabetes PNAS 99 3782 Walker L S K L J Ausubel A Chodos N Bekarian and A K Abbas 2002 CTLA
18. Specification Heat Dissipation One meter 3 ft of clearance on all sides for proper air flow If the laboratory containing the CyAn ADP is too small or the air conditioning not adequate to maintain 2 C the LP5 comes with 25 ft of hose that allows for the heat exchanger to be placed in an adjacent room The adjacent room must have thermal stability The water hoses to the heat exchanger cannot be greater than 50 ft long Other Options The cooling hoses can be hooked up to a variety of heat exchange systems or chillers The water system cooling the laser should be 10 35 C with stability of 1 8 CyAn ADP User Guide dakocytomation SECTION 3 SYSTEM OVERVIEW The CyAn ADP is a state of the art flow cytometer that utilizes one or more laser excitation sources to analyze biological cells beads or other microscopic particles as they are transported through an interrogation point in single file Information can be gathered from large numbers of particles in a relatively short time so that populations can be studied or differentiated from other populations using simple to complex statistical methods Physical and biochemical characteristics that can be measured using this instrument include but are not limited to forward scatter size related side scatter morphology related fluorescence from tagged stained cells particles and auto fluorescence from non stained cells particles CyAn ADP Feature
19. magnetic positive break E type interlock switch Operates dual spring solenoid shutter actuators on all laser paths Laser product class CLASS 1 LASER PRODUCT IEC EN 60825 1 A2 2001 Laser light leakage Conforms to 21CFR1040 10 and 21CFR1040 11 Operating Environment Ambient temperature 15 to 30 C 59 to 86 F For optimum performance maintain at 2 C Relative humidity 20 to 80 RH non condensing CyAn ADP System Utility Requirements amp Fusing Power UV Model not including Enterprise Il 621 Laser 115 VAC 10 60 Hz single phase 1 5A Power standard ADP 115 VAC 10 60 Hz single phase 1 75A Fuse Type 5x20mm low breaking high capacity IEC 115 VAC operation 6 3A Power Sheath Management System 100 240 VAC 50 60 Hz single phase 2A Fuse Sheath Management System Type 5x20mm low breaking high capacity UL 115 VAC operation 3A 130 CyAn ADP User Guide JOB Power Summit Workstation not fused 115 VAC 10 60 Hz single phase 1A Power Summit Workstation Monitor not fused 115 VAC 10 60 Hz single phase 2A Power External Transformer Input 230 VAC 10 50 60 Hz 5A Output 15 VAC 10 60 Hz 8A Fuse External Transformer power Type 5x20mm low breaking high capacity IEC 115 VAC operation 6 3A CyAn ADP UV Model Laser System Utility R
20. 05 114 649 127 11 OP 4 22 15115 10000 Dr 15115 10000 0 Qw le D naQ o oo o ooo 14876 9842 Ka 14833 9813 23 158 232 1 87 11 14 1 38 CyAn ADP User Guide 73 After creating the necessary histograms and quad regions gate the lymphocyte population from region R1 to the 4 dot plots PS E3 G1 R1 Non Stained Loi 2008 100 00 D Qoo 0 00 0 00 o noo 000 0 00 2078 100 00 D 000 2008 100 00 0 00 o Q00 2008 100 00 U Q00 74 CyAn ADP User Guide Ge DakoCytomation Run the non stained sample If necessary use the PMT controls to set the negative population for each fluorescent channel within the first log decade CyAn ADP User Guide 2008 100 00 5 1 74 4 Oa 1965 97 66 4 0a 2008 100 00 D om 0 oo z000 3960 H 040 323 379 4 37 11 14 13 3 10 75 3 23 379 0 00 000 Dm 000 3 23 205 1 5 213 2008 10000 11 n55 o odo 1933 9875 i4 070 32 2 55 3 40 11 14 000 0 00 328 246 Total R14 R15 R16 Ai 2008 10000 D Qao o Q00 1934 93 30 14 A0 32 1 33 O00 0 00 000 0 00 328 1 33 10 37 1 38 75 Running the First Sample After gating the dot plots for the lymphocyte population within region R1 run the first single color sample In theory it does not matter which of the 5 single color samples you begin with although in this example we are starting with FITC When the first single col
21. APC versus PE Cy5 and APC versus FITC the helper cells will appear in the right upper quadrant of all plots indicating a positive signal for both parameters I G1 R1 All Stains Pie E3 f1 G1 R1 All Stains v G1 RIJ All Stains PE C y5 Comp T T on 10 10 10 10 10 10 FITC Coma APC Comp Helper subset of the T cells Helper subset of the T cells Helper subset of the T cells CD45 PE Cy5 CD4S PE Cy5 CD45 PE Cy5 CD3 IIC CD3 FITC cD3 ITO CD APC CD8t APC CDS APC Suppressor subset of the T cell subset of white cells Suppressor cells a subset of the T cell population of white cells are CD45 PE Cy5 and CD3 FITC and CD8 APC Cy7 Using the all stains sample when 3 dot plots are created with compensated FITC versus PE Cy5 APC Cy7 versus PE Cy5 and APC Cy7 versus FITC the suppressor cells will appear within the right upper quadrant of all plots indicating a positive signal for both parameters L G1 R1 All Stains P ES G1 R1 All Stains L RII All Stains R24 Ras 104 10 T T 10 10 10 10 104 ABC CT Corte 101 1103 10t FITC Coma Suppressor subset of the T cells CD45 FE Cy5 Char FITC CDR APC Cy7 APC CyT COTA Suppressor subset of the T cells CD45 PE Cy5 cD3 MTC CO WP Ce Suppressor subset of the T cells CD45 PE Cy5 cb3 Itc CDS APC Cy
22. Density contour method Histogram Properties General Iw Enable Contours Smoothing Resolution Axis Contour Methods Equal Probability D 64 a Axis Ticks Dot Plot Display None D C 128 wn None Display Display A Contours Outliers Data Resolution Levels Il Events hy Max Threshold 11 im a Step Size 10 50 Display All Events One option in Summit that deserves special mention is the ability to simultaneously display data using both a contour and density plot To activate this feature use the dot plot display drop down menu and select Display all events The data will then be displayed as a density plot overlayed with contour lines based on the selected contouring method Below shows a density plot with Equal Probability contouring applied EI Contour Example 104 CyAn ADP User Guide 97 Data Resolution The Analog Particle or Cell Signal Cells or particles are suspended in a fluid medium and passed though the cytometer Carried by the stream the individual cells or particles pass sequentially through specific laser illumination zones At these interrogation points information is obtained on each cell including the real time detection of reflected and refracted light and laser stimulated emission of the fluorescent markers Laser H n Emitted H Q Signal Interrogation Q Point The emitted light and fluorescent signals are then passed to detectors or photomltiplier tubes
23. M Klempt and C Sorg 2000 Transendothelial migration of 27E10 human monocytes Int Immunol 12 1593 Qu C T M Moran and G J Randolph 2003 Autocrine type IFN and contact with endothelium promote the presentation of influenza A virus by monocyte derived APC J Immunol 170 1010 Wang Y C G Kelly J T Karttunen T Whittail P J Lehner L Duncan P MacAry J S Younson M Singh W Oehlmann G Cheng L Bergmeier and T Lehner 2001 CD40 is a cellular receptor mediating mycobacterial heat shock protein 70 stimulation of CC chemokines Immunity 15 971 983 NK Cells Blanca I R E W Bere HA Young and J R Ortaldo 2001 Human B cell activation by autologous NK cells is regulated by CD40 CD40 ligand interaction role of memory B cells and CD5 B cells J Immunol 167 11 6132 6139 Carayannopoulos L N O V Naidenko D H Fremont and W M Yokoyama 2002 Cutting edge Murine UL16 binding protein like transcript 1 A newly described transcript encoding a high affinity ligand for murine NKG2D J Immunol 169 4079 Gosselin P L H Mason J Willette Brown J R Ortaldo D W McVicar and S K Anderson 1999 Induction of DAP12 phosphorylation calcium mobilization and cytokine secretion by Ly49H J Leukocyte Biol 66 1 165 1771 Hoshino T R H Wiltrout and H A Young 1999 IL 18 is a potent coinducer of IL 13 in NK and T cells A new potential role for IL 18 in modulating the immune response J Immunol 162 5
24. PE FITC Comp E coun 2 Tctal R2 R3 R4 CyAn ADP User Guide 2453 T0000 0 om 0 om z165 888 oo om 88 34 Med 2 64 316 oo 0 00 0 00 000 237 305 0 00 0 00 00 000 3 2 205 11 06 23714 205 10 VIN EK gue Total 2433 10000 RE 0 goo R7 0 op AB 2165 8898 Tatal 2433 100 00 R14 0 ooo R15 o Da0 Alb 2164 8894 Riz 29 11 06 264 1 29 0 00 0 00 O00 0 00 237 1 38 79 Why adjust compensation for the PE versus FITC plot when FITC versus PE was compensated for the FITC sample This is because the fluorescent spillover occurs both ways FITC into the PE channel and PE into the FITC channel Look at the spectral emission curves for both fluorochromes you will notice that the degree of spillover between the two fluorochromes actually differs This is why compensation must be adjusted based on both the FITC and PE single color samples Emission Spectra 502 552 Wavelength nm 80 CyAn ADP User Guide dakocytomation Running the Remaining Samples Now run the third single color sample in this case PE Cy5 Again remember to reset the parameters along the dot plot axes so that PE Cy5 is plotted against the other 4 colors PE Cy5 vs FITC PE Cy5 vs PE PE Cy5 vs APC and PE Cy5 vs APC Cy7 Note these steps will be repeated for the remaining single color samples G1 R1 PE Cy5 E3 G1 R1 PE Cy5 v PE Cy5 T 1879 1000 35227 4 0 om 0 om 5
25. Scale Smoothing Initial Range 35 E Axis Axis Ticks Increment by 50 2 Univariates Set Fixed Scale MO Fix Scale Bin Resolution Auto Scale Hit Value 30 Increment by so H Ignore Lower P a Channels Ignore Upper bk Channels X close Manual Scaling Data can be manually re scaled in any histogram Simply select the histogram of interest and click the or icon Manual Scaling Options There are also a number of settings that can be adjusted for manually scaling the data Manual Scale Initial Range 35 Increment by 50 H m Set Fixed Scale MO M Fix Scale Increment By The Increment by value determines the degree to which the Y axis scale is changed when histogram data is manually re scaled using either the or icon For example suppose the manual scale increment value is set to 50 When histogram data is re scaled the Y axis value will increment by a factor of 50 or 1 2 depending on where the data is scaled up or down Below shows a histogram with the Y axis set to 1000 counts CyAn ADP User Guide 109 B Data Scaling Example 1000 750 When the tt icon is clicked the histogram data is manually reduced in size in the histogram This increases the Y axis scale by 50 in this case from 1000 to 1500 to reflect the visual scaling down of the data When the icon is clicked the data increases in size in the histogram As the data is scaled up the count valu
26. The DakoCytomation instrument product line conforms to international regulations encompassing the accessibility of high voltages by the user In the United States each flow cytometer is manufactured to comply with applicable regulations from the American National Standards Institute ANSI the Occupational Safety and Health Administration OSHA and various state regulatory organizations Internationally each CyAn ADP High Performance Flow Cytometer is manufactured to comply with the International Electrotechnical Commission s Standard for Safety Requirements for Electrical Equipment for Measurement Control and Laboratory Use IEC 61010 1 and subsequent amendments Biohazard Safety The CyAn ADP instrument may be used for the analysis of pathogenic or other harmful agents These operations can constitute a biohazard for the operator as well as bystanders in the surrounding laboratory space DakoCytomation does not certify any instrumentation for use with any hazardous organism or agent DakoCytomation strongly recommends explicit guidance be obtained from the institutional or company biosafety officer prior to operating this instrument with any potentially hazardous organism or agent WARNING If any biohazardous organism or agent is used in this instrument the user must inform DakoCytomation in writing prior to any field service visit or the return of any part to DakoCytomation or its vendors for service Safety of the CyAn ADP user
27. The calculated positives in this case are not likely to represent true positives Subtraction FL 2 700 Control CyAn ADP User Guide 121 The Overton method helps to minimize these false positives that are reported within the lower channels Again this is done by calculating the positive events based on the control events within the equivalent and all higher channels Ei Subtraction FL 2 700 Control Sample 4 4 wy Positives 122 CyAn ADP User Guide OR APPENDIX A CYAN ADP CONSUMABLES Table A 1 Calibration Particles Code Product KO111 SpectrAlign 3 0 um 1 x 10 mL 2 mL vial Single population of beads exciting and emitting across 351 nm 405 nm 488 nm and 635 nm K0110 FluoroSpheres 6 peak 3 2 um 1 x 10 mL 2 mL vial K0112 FluoroSpheres 8 peak 3 0 um 1 x 10 mL 5 mL vial K0113 SpectraComp Kit 1 Blank FITC PE PE Cy5 3 2 um 1 x 10 mL 1 mL vial K0114 SpectraComp Kit 2 Blank FITC PE PE Cy5 amp APC 3 2 um 1 x 10 mL 1 mL vial K0115 SpectraComp Kit 3 Blank FITC PE PE TR PE Cy5 3 2 um 1 x 10 mL 1 mL vial K0116 SpectraComp Kit 4 Blank FITC PE PE TR PE Cy5 PE Cy7 APC A APC Cy7 3 2 um 1 x 10 mL 1 mL vial K0117 SpectraComp Blank Particles 5 mL vial K0118 SpectraComp FITC Particles 1mL vial K0125 SpectraComp PE Particles 1mL vial K0126 SpectraComp PE
28. These components include trigger and signal processing and multi function input output Peripheral to the electronic chassis but within the instrument are a number of devices that can be grouped as fluidics control and sense pumps regulators valves and sensors laser shutter control and sense PMT voltage control and PMT signal The multifunction I O card is used for controlling these peripheral devices and sensing instrument status The PMT and photodiode detectors convert light emitted from particles excited at the interrogation point into electrical signals These signals are input to the trigger and signal processing cards Amplification analog to digital conversion and sampling techniques provide quantitative measurements including peak area integral log and pulse width for a given triggered particle event Peripheral Devices The CyAn ADP instrument has the following peripheral devices Summit Workstation two Monitors a printer and Sheath Management System SMS The SMS houses sheath container waste container cleaner cubitainer level indicators in line sheath filter and compressor vacuum pump Software CyAn ADP uses Summit Data Acquisition and Analysis Software for instrument control data acquisition and subsequent data analysis Summit is the Windows user interface for the entire DakoCytomation flow cytometry product line Summit software offers users complete control of the CyAn ADP instrument at varying levels o
29. User Guide User Resources For the latest information on DakoCytomation products and services please visit the DakoCytomation Web site at http Awww dakocytomation com Scope This guide provides a detailed discussion of the architecture and operating procedures for the CyAn ADP High Performance Flow Cytometer Detailed operating instructions for Summit software can be found in the Summit online help system The information contained in this document can be applied to all CyAn products Disclaimers This document is not a substitute for the detailed operator training provided by DakoCytomation or for other advanced instruction in general cytometric techniques It is essential that the operator have a working knowledge of Microsoft Windows XP or current Operating System prior to using this guide DakoCytomation also recommends the use of all of the networking services provided in the individual operator s laboratory Although daily maintenance and routine instrument adjustments are discussed here your local DakoCytomation Technical Service Group should be contacted immediately for assistance in the event of any instrument malfunction Trademarks Microsoft and Windows XP are registered trademarks of Microsoft Corporation Macintosh is a registered trademark of Apple Inc MoFlo is a registered trademark of DakoCytomation CyAn Summit SpectrAlign and SpectraComp are trademarks of DakoCytomation All other tr
30. a percentage from an unknown If we merely relied upon the measured value of a marker with an unknown percentage of cross contamination then we deliberately introduce an unknown degree of error into the result However this is entirely unnecessary We can empirically compute the amount of cross contamination indeed that s the purpose of compensation and we can subtract a known value from a true value thus eliminating this error Failure to use the true values as operands in calculating compensation results in population spread so clearly documented in the literature and introduction of mathematically anomalous sub populations that never existed in the real world Consider the following example An arbitrary range exists from 0 no fluorescence to 1024 bright beyond our detector means positive for that marker measure 500 means negative for that marker measure lt 500 Instrument is aligned for the negative population at 100 Instrument is aligned for the positive population at 1000 The spillover from PE to the FITC measure is 5 of the true PE The spillover from FITC to the PE measure is 30 of the true FITC Calculated spillover is the spillover constant times the fluorescent value above negative above 100 Since the instrument s PMTs are arbitrarily aligned to negative populations at 100 a population negative for all markers should have all markers reporting that populati
31. along with the ensuing scatter and fluorescence are directed within the optical system Electronics are used to power and control the instrument functions The software provides the user interface for the above fluidics optical 10 CyAn ADP User Guide and electronic hardware and the functions to acquire analyze and store data associated with the particles Fluidics The Sheath Management System controls the transfer of sheath fluid waste and cleaner fluid throughout the CyAn ADP system Sheath fluid is housed in either a replaceable 20 liter cubitainer or 20 liter plastic carboy Waste is contained in a 20 liter plastic carboy Cleaner fluid is contained in a 5 liter cubitainer The Sheath Management System provides improved sheath pressure stability and prevents bubbles from entering the system when the sheath container is changed The built in cleaner fluid cubitainer allows the user to easily clean and rinse the sheath path using DakoCytomation Clean and Rinse solution and sheath or DI water as rinsing fluid The 20 liter sheath and waste containers provide approximately 24 hours of system run time Figure 3 2 outlines the key fluidics components of CyAn ADP In general flow of air and fluids follows a path left to right in the diagram An air compressor provides pressure for propulsion of the sample cleaner and sheath fluids Air regulators condition and stabilize the pressure source prior to sheath and cleaner reservoirs and sample v
32. and F Mackay 2000 BAFF mediates survival of peripheral immature B lymphocytes J Exp Med 192 10 1453 Baumgarth N G C Jager O C Herman L A Herzenberg and L A Herzenberg 2000 CD4 T cells derived from B cell deficient mice inhibit the establishment of peripheral B cell pools PNAS 97 9 4766 CyAn ADP User Guide 133 Baumgarth N O C Herman G C Jager L E Brown L A Herzenberg and J Chen 2000 B 1 and B 2 cell derived immunoglobulin M antibodies are nonredundant components of the protective response to influenza virus infection J Exp Med 192 2 271 280 Benschop R J K Aviszus S Zhang T Manser J C Cambier and L J Wysocki 2001 Activation and anergy in bone marrow B cells of a novel immunoglobulin transgenic mouse that is both hapten specific and autoreactive Immunity 14 1 33 43 Bross L Y Fukita F McBlane C Demolliere K Rajewsky and H Jacobs 2000 DNA double strand breaks in immunoglobulin genes undergoing somatic hypermutation Immunity 13 589 Bross L M Muramatsu K Kinoshita T Honjo and H Jacobs 2002 DNA double strand breaks Prior to but not sufficient in targeting hypermutation J Exp Med 195 1187 Clarke S H and L W Arnold 1998 B 1 cell development evidence for an uncommitted immunoglobulin Ig M B cell precursor in B 1 cell differentiation J Exp Med 187 1325 Davis R S H Li C C Chen Y H Wang M D Cooper and P D Burrows 2002 Definition of a
33. and we use the indicated bands for their respective measures Bandpass Filter 530 30 ae Bandpass Filter 585 42 Emission Spectra Bandpass Filter 682 33 Wavelength nm The spillover from PE to the FITC measure is 5 of the true PE The spillover from Cy5 PE to the FITC measure is 1 of the true Cy5 PE The spillover from FITC to the PE measure is 30 of the true FITC The spillover from Cy5 PE to the PE measure is 25 of the true Cy5 PE The spillover from FITC to the Cy5 PE measure is 0 of the true FITC The spillover from PE to the Cy5 PE measure is 8 of the true PE This yields the following compensation matrix FITC PE Cy5 PE FITC n a 5 00 1 00 PE 30 00 n a 25 00 Cy5 PE 0 00 8 00 n a 68 CyAn ADP User Guide vakocytomation The true and measured values are Bandpass Filter 530 30 Bandpass Filter 585 42 Emission Spectra Bandpass Filter 682 33 Wavelength nm FITC true a FITC measured a b f PE true c PE measured c d g Cy5 PE true e Cy5 PE measured e i h Please note that for completeness we include h in our calculations but we state in this example that there is no spillover from FITC to Cy5 PE represented by the area of h Assuming perfect expression the following particles represent all possibilities for FITC and PE and Cy5 PE expression within a population Particle FITC PE Cy5 PEFITCmeasure FITCtrue PEmeasure PEtru
34. automatically available under each histogram or may be detached for placement anywhere on the desktop Summit s Real world Desktop With Summit s real world desktop the user doesn t have to remember to load and save protocols Summit remembers everything the user does in a given session and the next time Summit is opened the desktop appears exactly as the user left it Every window color size and placement is the same as it was at the end of your last session No saving is required With Summit software you focus on the science CyAn ADP User Guide 19 20 CyAn ADP User Guide Gi DakoCytomation SECTION A STARTUP AND SHUTDOWN PROCEDURES Startup Procedure Required Reagents DakoCytomation Decontamination Solution catalog 82324 DakoCytomation Clean and Rinse Solution catalog 2323 CyAn ADP Startup Note The Quick Start Guides for the CyAn ADP Document Numbers 0000023 and 0000022 are available for a quick reference for Startup and Shutdown procedures CyAn ADP UV Model users Turn on the power to the Laser Power Supply by turning the key to the On position and turning the switch to the On position All CyAn ADP users 1 Log on to the Summit workstation 2 Open Summit by double clicking the Summit icon on the Windows desktop 3 Select an existing database or create a new one and then click OK Summit 4 1 E Select database c Program Files D akoCytomationS ummit 4 1 Listmodetest sum a
35. but the control and sample designations are switched The outcome is a different positive population identified within the two histograms 114 CyAn ADP User Guide dakocytomation B Subtraction FL2 1240 9304 Control Positives Analysis samples within a subtraction histogram including the designated control can be seen within the histogram s legend To access this list right click within the subtraction histogram and select Show Legend from the popup menu E Subtraction FL 2 2046 ch Subtraction Method X gt Posit FT New Bar Region Co fat Add Histogram Data Set Fixed Normalization wi Auto Show Statistics Properties The legend will contain a list of data sets associated with the subtraction histogram with the designated control displayed in bold type By default the first data set added to a subtraction CyAn ADP User Guide 115 histogram is designated as the control Events for this data are then subtracted from all subsequently added samples to determine any positive events If necessary the sample designated as the control can be changed by right clicking on a different sample and selecting Control from the popup menu Sec HEr Histogram Experiment ra Histogram Experiment H Control Analysis H Control Analysis Sample Analysis P is Positives Positives Positives F Properties L Delete Subtraction Methods Summit provides a coupl
36. cm wide x 45 cm deep x 40 cm high and the CyAn UV Model measures 120 cm wide x 60 cm deep x 40 cm high Summit Software Summit Software DakoCytomation s premier cytometry software product offers full control of all CyAn ADP functions coupled with robust user and application specific data acquisition analysis storage reduction and retrieval Summit controls all instrument parameters in an_ intuitive MS CyAn ADP User Guide 9 Windows based drag and drop environment using simple protocol based menus Summit also provides high content data acquisition database capabilities and open architecture for data exchange Additional features include off line analysis and easy page layout Safety The CyAn ADP High Performance Cell Bead Analyzer has been designed to incorporate the highest level of laboratory and operator safety CyAn ADP Subsystems CyAn ADP has four key subsystems that form a powerful research tool The four subsystems are Fluidics Optics Electronics Software Fluidics Electronics Software Control amp Signal A D 5 Processing PC Electronics el D e Figure 3 1 CyAn ADP Functional Block Diagram A functional block diagram for the CyAn ADP High Performance Flow Cytometer is shown in figure 3 1 The fluidics system pressurizes the system and transports particles to the interrogation point Lasers are used as excitation sources and their beams
37. control thus it is assigned to a lower set of channels Using the Overton method these events are not reported as positive since the algorithm also accounts for the control events present within higher channels sample events per channel as a of control events per channel and all higher channels Subtraction FL 1 Sample No Positives Reported 120 CyAn ADP User Guide di tomation If the channel method were used in this case the following positive population would be calculated and reported However it is very unlikely that these events actually represent true positives Ei Subtraction FL 1 Sample Control Positives Another advantage of the Overton method is the reduction of false positives reported in the lower channels simply because more events were collected in those channels for a sample versus the control This is especially evident when using subtraction histograms to compare data sets with different numbers of total counts For example the following is a subtraction histogram where the control contains 50 000 total events but the sample contains 100 000 events Because the channel method reports positives based on the difference in the number of events between the sample and control on a per channel basis many channels will report positive events simply because more events were collected on the sample tube and thus are likely to predominate in number within certain channels than the control
38. digital and all the benefits of software compensation are maintained there only needs to be a hardware component that can make real time sort decisions based on other parameters without going through the possibly flawed multi dimensional re map from true to real space as is done with software compensation for sorting That s the job of DakoCytomation s Computed Parameter Board which uses a DSP processor Compensation is still performed in software and both the raw and compensated values can be saved to the data file for all the benefits of no data or precision loss However the hardware makes the exact same calculation that is made by the software so real time sort decisions are made with the ideal algorithm Advantages All the benefits of hardware compensation All the benefits of software compensation By removing restrictions in a new flexible hardware architecture and maintaining all the advantages of software manipulation the user can apply software compensation with hardware real time sort decisions to get the best of both worlds In fact the implication states that almost any computation can be made on a per event basis to decide whether or not an event is sorted This wasn t possible for the thirty years prior to DSP technology being released in 1999 Of course if you don t need to sort you simply analyze then software compensation is probably what you are after and the DSP solution providing you real time comp
39. exists between the two should a given particle express both markers Emission Spectra 502 552 Wavelength nm One of the reasons these markers are used together is that each is adequately excited by a 488nm laser For example many flow cytometers have one or two lasers but two or three detectors may exist on each laser path If many markers can be sufficiently excited by a single laser but fluoresce in different wavelengths they can be properly resolved on relatively inexpensive cytometers because only one laser is needed for multiple markers Below is the relative excitation efficiencies for FITC and PE including a line identifying the commonly used 488nm laser line used to excite these markers CyAn ADP User Guide 43 Excitation Spectra 502 552 Wavelength nm As seen on the graph each is excited at 488nm FITC at approximately 90 efficiency and PE at approximately 60 efficiency As previously shown FITC and PE will fluoresce at different spectra Therefore it is possible to resolve both FITC and PE even though each was excited by the same laser because of differences between their spectral emission properties Further as shown by comparing the emission graph with the excitation graph it is common for the excitation wavelength to be higher energy shorter wavelength than the fluorescence which is lower energy longer wavelength With the addition of filters we can reduce noise or cross contamination fr
40. is critically dependent upon interleukin 7 J Exp Med 196 5 705 711 Ohdan H K G Swenson H S Kruger Gray Y G Yang Y Xu A D Thall and M Sykes 2000 Mac 1 negative B 1b phenotype of natural antibody producing cells including those responding to Gala1 3Gal epitopes in a1 3 galactosyltransferase deficient mice J Immunol 165 5518 134 CyAn ADP User Guide Gi DakoCytomation Rolink A H T Brocker H Bluethmann M H Kosco Vilbois J Andersson and F Melchers 1999 Mutations affecting either generation of survival of cells influence the pool size of mature B cells mmunity 10 619 628 Rolink A H T Winkler F Melchers and J Anderson 2000 Precursor B cell receptor dependent B cell proliferation and differentiation does not require the bone marrow or fetal liver environment J Exp Med 191 1 23 31 Rossi M D T Yokota K L Medina K P Garrett P C Comp A H Schipul Jr and P W Kincade 2003 B lymphopoiesis is active throughout human life but there are developmental age related changes Blood 101 576 Sale J E and M S Neuberger 1998 TdT accessible breaks are scattered over the immunoglobulin V domain in a constitutively hypermutating B cell line Immunity 9 859 869 Seo W J M L Fields J L Buckler A J Reed L Mandik Nayak S A Nish R J Noelle L A Turka F D Finkelman A J Caton and J Erickson 2002 The impact of T helper and T regulatory cells on the regulation of anti dou
41. partially replaces EBNA2 function in B cells immortalized by Epstein Barr virus J Virol 75 5899 Johnson J A and J D Gangemi 1999 Selective inhibition of human papillomavirus induced cell proliferation by S 1 3 hydroxy 2 phosphonylmethoxy propyl cytosine Antimicrob Agents Chemother 43 1198 Joseph A M G J Babcock and D A Thorley Lawson 2000 Cells expressing the Epstein Barr virus growth program are present in and restricted to the naive B cell subset of healthy tonsils J Virol 74 9964 Koehne G H F Gallardo M Sadelain and R J O Reilly 2000 Rapid selection of antigen specific T lymphocytes by retroviral transduction Blood 96 109 Koehne G K M Smith T L Ferguson R Y Williams G Heller E G Pamer B Dupont and R J O Reilly 2002 Quantitation selection and functional characterization of Epstein Barr virus specific and alloreactive T cells detected by intracellular interferon y production and growth of cytotoxic precursors Blood 99 1730 Kuehnle l M H Huls Z Liu M Semmelmann R A Krance M K Brenner C M Rooney and H E Heslop 2000 CD20 monoclonal antibody rituximab for therapy of Epstein Barr virus lymphoma after hemopoietic stem cell transplantation Blood 95 1502 Laichalk L L D Hochberg G J Babcock R B Freeman and D A Thorley Lawson 2002 The dispersal of mucosal memory B cells Evidence from persistent EBV infection Immunity 16 745 Leon R P T
42. positive and negative populations G1 R1 APC Pie E3 G1 R1 APC BS st 3 16 305 0 nw oO 000 RB o Q00 0 Dm 00 000 A 0 Qao 437 DAD 2 205 AB 4371 6282 2357 365 2567 37 18 67 32 205 AS 2537 37 18 67 52 3 52 l G1 RI APC M E3 pic G1 RI APC 104 S 3 PE CyS Comp B APC Cy Comp e958 10000 316 1 00 3 16 1 00 0 Dm 00 O00 0 ooo 00 0 00 0 Dm OM Q00 0 Q00 O00 0 00 4371 62 amp 2 37 1 00 431 6292 237 1 00 2567 3718 GH 1 00 2597 _ 37 18 67 32 1 00 84 CyAn ADP User Guide Gi DakoCytomation Run the 5th and final single color sample APC Cy7 Pair the APC Cy7 parameter against FITC PE PE Cy5 and APC for the dot plots G4 R1 APC Cy7 Ell mi R1 APC Cy 324 100 00 1 002 D nao 3605 67 71 1718 3227 1718 3227 G1 RI APC Cy7 PE CyS Comp o oo 1 002 d 2 6773 170 3227 1717 22285 CyAn ADP User Guide 85 Where necessary adjust compensation to align the median fluorescence relative to the other 4 colors between the APC positive and negative populations Notice that for this sample compensation only needed to be adjusted for the APC Cy7 versus APC plot G1 R1 APC Cy7 PS ES G1 R1 APC Cy Oo x 104 10 10 1 VIN 10t APC CyT Taal SA 1000 3 5304 100 00 3 0 00 D 1 DON o om o o op em 67 73 36005 67 71 17183227 1718 3227 G1 R1 APC Cy7 104 S FE Cap Camp R
43. power on the LP5 is off and the Cool Down Cycle is on Note With the Cool Down Cycle switch on the LP5 unit will remain on until the laser has sufficiently cooled down 10 Close Summit Note DakoCytomation recommends waiting 30 seconds after closing Summit before logging off of the workstation in order to allow all data sources to close 11 Log off of the workstation and turn off the computer 12 Turn off the computer monitor s 26 CyAn ADP User Guide Mos oCytomation SECTION 5 FLUIDS MANAGEMENT Sheath Management System Indicators The Sheath Management System provides 20 24 hours of sample run time As the level of sheath fluid decreases and the level of waste fluid increases both Summit software and Light Emitting Diodes LED indicators on the Sheath Management System front panel will provide fluid level status information and alerts to the user SMS indicators can be viewed in the Sheath Management System section of the Instrument tab in the Control Panel There are nine indicator lights in the SMS area When all nine indicator lights are green all components of the SMS are functioning properly ie Ble lowell JF Ca i me instrument Acquisition Sample Histogram Gating Workspace Event rate 488nm Opened MV On All 100 351nm ime Opened Boost On 635nm mmm Opened i Startup Shutdown Backflush Vacuum bp Low Lever Debubble Clean
44. region around the suspected doublets 2 Right click in the region and then click Select Gate Color from the menu 3 If necessary create other regions to identify doublets 4 Move the regions around to help identify the doublet population 5 Set your sort decisions so that doublets are eliminated from the sort Below are some histograms that show an example of color gating in Summit This data was gathered by analyzing a bead lot that had been centrifuged to create a preponderance of doublets They are spherical beads with one size and one fluorescent intensity In a perfect world with no doublets we would see one population in scatter and fluorescence plots Instead we see the doublets shown below in purple 106 CyAn ADP User Guide Gi DakoCytomation Color Gating 256 Pulse Width 3924 2943 1962 981 0 0 128 Pulse Width Methods of Doublet Discrimination There are many ways to perform doublet discrimination that involve integrated signals and the pulse width parameter In fact it s possible and often helpful to combine two or more means through gating on multiple histograms Below are some of the more common methods FSC Area versus Pulse Width FSC ADC is set to Area Create FSC area versus PW histogram in Summit FSC Area versus SSC Integrated Both FSC and SSC ADC s are set to Area FLX Area versus FLX Peak Change BNC wiring to enable the same parameter to be processed on two different ADC s
45. reported under the green sample curve Ei Subtraction FL 1 Sample Control No Positives Reported CyAn ADP User Guide 119 Experimental and Biological Relevance The impact of using the Overton method for analyzing experimental data is demonstrated by the following example Suppose data is first collected on a true FlTC negative control followed by a potentially FITC positive sample Data for the two samples is then added to a subtraction histogram to identify any positive events Keep in mind that the events displayed within the histogram are binned and assigned to a channel based on an event s recorded intensity value for a particular parameter in this case FITC Thus for true FITC positive events one would expect a shift in the data to the right or into a higher grouping of channels i e a stronger signal is recorded from any FITC positive cells and subsequently those events are assigned into higher channels within the histogram With the Overton method sample events falling upstream of the control are reported as positives Ei Subtraction FL 1 Negative Control Expected Positives However take a situation where a potential FITC positive sample is analyzed and some or all of the data falls equal to or below the negative control From an experimental perspective it is unlikely that the sample is actually FITC positive since the recorded FITC signal is equivalent to or weaker than the non FITC
46. the advantage of a having a higher channel number within a histogram or dot plot is better resolution of the data This means that a group of events that were indistinguishable from one another because they were binned within the same channel are now distributed as subgroups within several channels Increased resolution is useful when distinguishing between populations with similar signal intensities e g chromosomes The major drawback to having an increased number of channels is that many more events must be collected for the sample to achieve nice Gaussian curves or peaks within the data versus having a flat data profile DI Resolution Example 0 1024 2046 3072 4096 FSC Bega Cout Hist A Total 50000 100 00 100 00 lt gt CyAn ADP User Guide 103 When increasing the resolution within dot plots the data will become more disperse due to the greater number of channels along both the X and Y axis When decreasing the resolution within dot plots the data will become intensified due to the reduced number of channels along the X and Y axis Both of these effects can be seen in the set of dot plots below Resolution Example K 256 Ei Resolution Example 1024 192 128 DS 64 768 B Resolution Example 256 64 192 CH 1128 L 64 104 CyAn ADP User Guide pexocytomation Doublet Discrimination Doublets are an inevitable element in flow cytometry The probabi
47. we might want to use compensation to manipulate the observed responses to be more in line with what we know to be truly biologically significant Using the compensation matrix we defined above the value for computing the compensated values are the following Yellow comp Yellow 0 3 Green 0 0 Blue Green comp Green 0 3 Yellow 0 3 Blue Blue comp Blue 0 0 Yellow 0 3 Green For this example we only want to compute Yellow The results are the same if you compute Green or Blue Doing the math for Yellow compensation on each of our events we get the following 1 Yellow comp 0 0 3 O Green 0 0 O Blue 0 0 2 Yellow comp 10 0 3 3 Green 0 0 O Blue 9 1 3 Yellow comp 3 0 3 10 Green 0 0 3 Blue 0 0 4 Yellow comp 0 0 3 3 Green 0 0 10 Blue 0 9 5 Yellow comp 10 0 3 6 Green 0 0 10 Blue 8 2 6 Yellow comp 13 0 3 13 Green 0 0 3 Blue 7 Yellow comp 3 0 3 13 Green 0 0 13 Blue 0 9 8 Yellow comp 13 0 3 16 Green 0 0 13 Blue 8 2 Remember that events 2 5 6 and 8 were positive for Yellow but the other events were negative for Yellow However we re starting to get some strange results some spreading from our calculations For example both events 2 and 5 had the same originally observed value 10 but after compensation they have diff
48. 0 0 0 10 0 3 6 10 6 Yellow comp 13 0 3 13 0 3 13 0 3 3 0 0 3 0 0 13 0 3 13 10 54 7 Yellow comp 3 0 3 13 0 3 3 0 3 13 0 0 13 0 0 3 0 3 13 0 54 8 Yellow comp 13 0 3 16 0 3 13 0 3 13 0 0 13 0 0 13 0 3 16 10 54 The user may notice that we are getting negative values of 0 0 and 0 54 in this example and positive values of 10 and 10 54 as opposed to negative compensation yielding negative values of 0 and 0 27 and positive values of 9 1 and 9 37 However we have actually performed the same mathematical manipulation but with the anomaly that the scale is compressed for negative compensation The range and spread in fact the validity of the result is actually identical when you perform the mathematical proofs This clears up the two disconcerting points for some researchers The user doesn t see negative constants Compensation is calculated only from the real signal not the artificially inflated signal The Blue color is still not directly considered when compensating against Yellow and correctly drops out of the equation Additionally the researcher only needs to identify the constants for colors with actual spectral overlap in this case one constant instead of constants for every color that may indirectly affect compensation in this case two constants T
49. 070 5077 Hoshino T R T Winkler Pickett A T Mason J R Ortaldo and H A Young 1999 IL 13 production by NK cells IL 13 producing NK and T cells are present in vivo in the absence of IFN y J Immunol 162 51 59 Makrigiannis A P J Etzler R Winkler Pickett A Mason J R Ortaldo and S K Anderson 2000 Identification of the Ly49L protein evidence for activating counterparts to inhibitory Ly49 proteins J Leukocyte Biol 68 5 765 771 CyAn ADP User Guide 139 Makrigiannis A P A T Pau A Saleh R Winkler Pickett J R Ortaldo and S K Anderson 2001 Class MHC binding characteristics of the 129 J Ly49 repertoire J Immunol 166 8 5034 5043 Mason L H J Willette Brown A T Mason D McVicar and J R Ortaldo 2000 Interaction of Ly 49D NK cells with H 2D target cells leads to Dap 12 phosphorylation and IFN y secretion J Immunol 164 603 611 McVicar D W R Winkler Pickett L S Taylor A Makrigiannis M Bennett S K Anderson and J R Ortaldo 2002 Aberrant DAP12 signaling in the 129 strain of mice Implications for the analysis of gene targeted mice J mmunol 169 1721 Orange J S N Ramesh E Remold O Donnell Y Sasahara L Koopman M Byrne F A Bonilla F S Rosen R S Geha and J L Strominger 2002 Wiskott Aldrich syndrome protein is required for NK cell cytotoxicity and colocalizes with actin to NK cell activating immunologic synapses PNAS 99 11351 Ortaldo J R E W Bere
50. 13 0 3 16 Green 0 09 13 Blue 9 37 By adding back some of the Blue that we over subtracted from Yellow we are getting better approximations Events 2 and 5 both move from values of 10 to values of 9 1 and events 6 and 8 both move from values of 13 to values of 9 37 Similarly events 1 and 4 both keep their original values of zero 4 does not become negative and events 3 and 7 both move from values of 3 to values of 0 27 Thus spreading as a result of compensation is removed Negative compensation may be a viable way to improve the compensation of data This brings up a couple of points Negative coefficients look strange At first glance it looks like something that shouldn t be done Many experienced researchers and operators even those that believe in compensation don t like the idea that these coefficients are negative We now have a non zero constant for the Blue color in our equation to calculate the Yellow compensated value where the Blue value should have dropped out of the equation after all we stated from a physics perspective that Blue and Yellow do not spectrally overlap so Blue should never be considered when calculating the compensated value for Yellow In essence we modify 50 CyAn ADP User Guide Gi Dako the proper equation with a work around constant to handle a mathematical anomaly It s not ideal but may be the best we can do to correct for errors introduced by analog compens
51. 3 Move on to the next fluorochrome PE by either running the PE stained only tube or by selecting the PE single stained data from the Data Navigator if the data has been previously stored in Summit Change the x axis on the histogram to reflect the PE data and change the data on the y axis to FITC Comp Optimize compensation for FITC Comp and then move on to PE TR Comp PE Cy5 Comp and so on 4 In general only fluorochromes with neighboring emission spectra need to be considered for compensation FITC and PE used in the same experiment require compensation for the FITC fluorescence in the PE channel FITC and Cy5 PE however do not overlap in their respective bandpass filter channels No compensation is required between these two channels Historical Compensation Calculation The problems with the historical compensation calculation include Homogeneous populations are mathematically divided into sub populations that do not biologically or physically exist Computation of the true value is arbitrary and based on per particle values of other markers Amplitude of the error is cumulative across additional colors and markers decreasing the sensitivity of experiments with additional markers Markers are mathematically arbitrarily correlated with markers that are physically unrelated Attempts to correct for the formulae deficiencies create additional ambiguities and error and may be difficult to explain e negative compensation com
52. 348 Gubbels M J C Li and B Striepen 2003 High throughput growth assay for Toxoplasma gondii using yellow fluorescent protein Antimicrob Agents Chemother 47 309 Kinsella T M C T Ohashi A G Harder G C Yam W Li B Peelle E S Pali M K Bennett S M Molineaux D A Anderson E S Masuda and D G Payan 2002 Retrovirally delivered random cyclic peptide libraries yield inhibitors of 138 CyAn ADP User Guide Gi DakoCytomation interleukin 4 signaling in human B cells J Biol Chem 277 37512 Lorens J B M K Bennett D M Pearsall W R Throndset A B Rossi R J Armstrong B P Fox E H Chan Y Luo E Masuda D A Ferrick D C Anderson D G Payan and G P Nolan 2000 Retroviral delivery of peptide modulators of cellular functions Molecular Therapy 1 5 438 447 Shires J E Theodoridis and A C Hayday 2001 Biological insights into TCRy8 and TCRaB intraepithelial lymphocytes provided by serial analysis of gene expression SAGE Immunity 15 419 Wehrman T B Kleaveland J Her R F Balint and H M Blau 2002 Protein protein interactions monitored in mammalian cells via complementation of B lactamase enzyme fragments PNAS 99 6 3469 3474 Wentzel A A Christmann T Adams and H Kolmar 2001 Display of passenger proteins on the surface of Escherichia coli K 12 by the enterohemorrhagic E coli intimin EaeA J Bacteriol 183 7273 Wentzel A A Christmann R Kratzner and H Kolm
53. 4 2 1072 1000 960 4 39 6 Differences between the computed and the true values are much more significant in this example beyond what was seen in the two color example For example even though the FITC and PE interaction remains constant the introduction of the Cy5 PE marker changes our error shift of 1 35 in our determination of the true value of PE from the two color example to as high as 3 42 for PE in the three color example more than doubling Further the contribution of both FITC and Cy5 PE to PE amplifies the same error resulting in a calculated error of 3 96 for Cy5 PE In other words too much PE is subtracted from Cy5 PE because that PE measure was arbitrarily inflated by both FITC and Cy5 PE Moreover the erroneous calculation essentially subtracts FITC from Cy5 PE even though we state that there is no physical association rather the historical calculation introduces a faulty mathematical association between FITC and Cy5 PE One anomaly deserves specific attention Population spreading While particles 5 6 7 and 8 all have the same true value the historical calculation striates that group into two sub populations One with a 1 8 error and one with a 3 96 error This is entirely a mathematical anomaly that creates population spreading or at high resolution population striation since these sub populations have a 2 16 shift from each other All three markers exhibit this shift for both positive and negative popu
54. 4 Differentially Regulates T cell responses to endogenous tissue protein versus exogenous immunogen J Immunol 169 6202 Wang B A Sharma R Maile M Saad E J Collins and J A Frelinger 2002 Peptidic termini play a significant role in TCR recognition J Immunol 169 3137 Wen R D Wang C McKay K D Bunting J C Marine E F Vanin G P Zambetti S J Korsmeyer J N Ihle and J L Cleveland 2001 Jak3 selectively regulates Bax and Bcl 2 expression to promote T cell development Mol Cell Biol 21 678 Wilson S B S C Kent H F Horton A A Hill P L Bollyky D A Hafler J L Strominger and M C Byrne 2000 Multiple differences in gene expression in regulatory Va 24JaQ T cells from identical twins discordant for type diabetes PNAS 97 7411 Winandy S L Wu J H Wang and K Georgopoulos 1999 Pre T cell receptor TCR and TCR controlled checkpoints in T cell differentiation are set by Ikaros J Exp Med 190 1039 Workman C J K J Dugger and D A A Vignali 2002 Cutting edge Molecular analysis of the negative regulatory function of lymphocyte activation gene 3 J mmunol 169 5392 Zhong X P E A Hainey B A Olenchock H Zhao M K Topham and G A Koretzky 2002 Regulation of T cell receptor induced activation of the ras ERK pathway by diacylglycerol kinase zeta J Biol Chem 277 31089 Viruses Babcock G J and D A Thorley Lawson 2000 Tonsillar memory B cells latently i
55. 7 CyAn ADP User Guide 89 To better identify the true suppressor cells a complex gating strategy can be used Because the suppressor cells should appear within the upper right quadrant of all three plots a gate can be set to include all three regions By applying this gate to all three plots only those cells that occur in all 3 regions will be displayed within each of the plots Additionally the gate for region R1 is also included Below illustrates how by applying a gate that includes regions R1 R25 R3 and R7 the suppressors can be separated from other lymphocyte cells L G8 R1 amp R25 amp R3 amp R7 All Stains PI EI G8 R1 amp R25 amp R3 amp R7 All Stains PI EJ 4 G8 R1 amp R25 amp R3 amp R7 All Stains PI EJ PE Cy5 Comp FITC Comp T T T T 0 T 1 102 10 10 10 10 10 401 1 10 104 FITC Log APC CyT Corte APC CyT Come Suppressor subset of the T cells Suppressor subset of the T cells Suppressor subset of the T cells cb4st PE Cys CD45 PE Cy5 CD45 PE Cy5 CO FITC cp3t FITC cp3 FITC cpa APC Cy7 CIR APC Cy7 CRT APC Cy7 White cells that are neither T or B cells i e NK cells Non T or B cells i e NK cells are CD45 PE Cy5 CD3 FITC and CD19 PE Using the all stains sample 3 plots can be created with compensated FITC versus PE Cy5 PE versus PE Cy5 and PE versus FITC In the FITC versus PE Cy5 and PE vers
56. 7 Perez O D G P Nolan D Magda R A Miller and L A Herzenberg 2002 Motexafin gadolinium Gd Tex selectively induces apoptosis in HIV 1 infected CD4 T helper cells PNAS 99 2270 Qu C T M Moran and G J Randolph 2003 Autocrine type IFN and contact with endothelium promote the presentation of influenza A virus by monocyte derived APC J Immunol 170 1010 Simard M C P Chrobak D G Kay Z Hanna S Jothy and P Jolicoeur 2002 Expression of simian immunodeficiency virus nef in immune cells of transgenic mice leads to a severe AIDS like disease J Virol 76 3981 Stripecke R A A Cardoso K A Pepper D C Skelton X J Yu L Mascarenhas K Weinberg L M Nadler and D B Kohn 2000 Lentiviral vectors for efficient delivery of CD80 and granulocyte macrophage colony stimulating factor in human acute lymphoblastic leukemia and acute myeloid leukemia cells to induce antileukemic immune responses Blood 96 1317 Sutkowski N B Conrad D A Thorley Lawson and B T Huber 2001 Epstein Barr virus transactivates the human endogenous retrovirus HERV K18 that encodes a superantigen Immunity 15 579 Szmania S A Galloway M Bruorton P Musk G Aubert A Arthur H Pyle N Hensel N Ta L Lamb Jr T Dodi A Madrigal J Barrett J Henslee Downey and F van Rhee 2001 Isolation and expansion of cytomegalovirus specific cytotoxic T lymphocytes to clinical scale from a single blood draw using
57. A mm 6S E 698 2 94 4540 9305 352 27 4 Sat 262 27 14 96 352 27 205 4879 100 00 0 00 000 0 n00 oo Q00 D O00 6 93 205 39 bon 352 27 205 4540 9205 CyAn ADP User Guide 81 Where necessary adjust compensation to align the median fluorescence relative to the other 4 colors between the PE Cy5 negative and positive populations 82 G1 R1 PE Cy5 FITC Comp 100 10 10 10 PE Cy5 Comp R2 o oO R3 D Qw R4 33 bn loj xi G1 R1 PE Cy5 104 10t 102 10 10 10 10t PE CyS 5 79 FATC 949 PE AB o ooo A 0 Qao AB z9 6695 Ag 4590 9205 R5 4540 33065 365 17 1 00 L G1 R1 PE Cy5 104 352 27 205 0 00 Q00 oo Q00 6 93 205 352 27 205 4879 100 00 SE o on o 000 9 695 4540 9305 CyAn ADP User Guide dakocytomation Run the 4th sample APC Pair the APC parameter against FITC PE PE Cy5 and APC Cy7 for the dot plots L G1 R1 APC 6958 10000 Tatal ESS T0000 316 3 65 0 om RE 0 Q00 Dm 0 00 D aw A7 0 QOO O00 0 00 437 62 A AB 4371 6282 237 3 65 287 37 18 Ag 2597 37 18 SI 3 52 L met Lk G1 RI APC 102 103 APC 316 KE Tatal 6938 10000 316 2 37 0 00 0 00 R14 1 00 O00 0 00 77 74 3398 R15 1729 2435 7234 35 23 2 37 246 R16 430 6291 23 1 65 62 64 1981 Ri 58 1233 5829 20 54 CyAn ADP User Guide 83 Where necessary adjust compensation to align the median fluorescence relative to the other 4 colors between the APC
58. Baker 1999 Characterization of differentially expressed genes in purified Drosophila follicle cells toward a general strategy for cell type specific developmental analysis PNAS 96 5559 Hipfner D R K Weigmann and S M Cohen 2002 The bantam gene regulates Drosophila growth Genetics 161 1527 Noureddine M A T D Donaldson S A Thacker and R J Duronio 2002 Drosophila Roc1a encodes a RING H2 protein with a unique function in processing the Hh signal transducer Ci by the SCF E3 ubiquitin ligase Dev Cell 2 757 Tapon N N Ito B J Dickson J E Treisman and K Hariharan 2001 The Drosophila tuberous sclerosis complex gene homologs restrict cell growth and cell proliferation Cell 105 345 Tseng A S K and I K Hariharan 2002 An overexpression screen in Drosophila for genes that restrict growth or cell cycle progression in the developing eye Genetics 162 229 Fluorescent Proteins Allenspach E J P Cullinan J Tong Q Tang A G Tesciuba J L Cannon S M Takahashi R Morgan J K Burkhardt and A I Sperling 2001 ERM dependent movement of CD43 defines a novel protein complex distal to the immunological synapse Immunity 15 739 750 Demo S D E Masuda A B Rossi B T Throndset A L Gerard E H Chan R J Armstrong B P Fox J b Lorens D G Pana R H Scheller and J M Fisher 1999 Quantitative measurement of mast cell degranulation using a novel flow cytometric annexin V binding assay C
59. Cy5 APC and APC Cy7 If FITC is the first single color to be run 4 72 CyAn ADP User Guide dakocytomation plots should be created FITC versus PE FITC versus PE Cy5 FITC versus APC and FITC versus APC Cy7 Typically we recommend that you plot FITC along the X axis and the other A colors along the Y If a 7 color experiment is being conducted 6 plots need to be created color 1 versus 2 1 versus 3 1 versus 4 1 versus 5 1 versus 6 and 1 versus 7 Additionally you will want to create a quadrant within each of the dot plots Statistics from the quad regions will be used in setting compensation from the control samples and identifying positive and negative populations in the all stains sample In general we recommend that you position the quadrants to encompass the 1st log decade Although this is not essential when setting and adjusting compensation when the all stains sample is run the quadrant will need to be aligned along the 1st decade mark to properly identify the positive and negative populations The graphic below shows 4 plots that have been created for the 5 color data Notice that FITC has been paired against the other 4 fluorochromes used in the experiment 1 Non Stained 108 Geh T T 100 101 102 10 FITC Total 15115 10000 1 84 3 05 15115 10000 1 64 2 13 R2 56 3d amp 49 11 97 113 075 583 1155 R3 148 OS 124 1433 62 D i 31 62 14 33 R4 14380 3500 1 78 294 14720 97 39 1 84 2 13 Ab fo
60. Cy5 Tube 5 CDA APC Tube 6 CD8 APC Cy7 Tube 7 All All Background When performing a multi color experiment on your instrument the forward and side light scatter parameters should be set to linear all fluorescent parameters should be set to log Create a forward versus side scatter dot plot and run the non stained sample This allows identification of the cell populations that will be used for gating Below is a dot plot showing unstained whole blood using both the forward and side scatter parameters The lymphocyte monocyte and granulocyte populations have been color coded for easy recognition CyAn ADP User Guide 71 JL Non Stained OF x 256 Region Count zl veel Total 15115 100 00 19 00 6 00 Within the forward side scatter plot create a region around the lymphocyte population This region will be used to gate the lymphocytes into additional histograms l gt Nep Stained 255 F5C Regin Goal z Medan Tota 15115 100 00 13 00 6 00 Al 208 1322 97 00 15 00 Tcal 15115 100 00 19 00 600 Setting Up Histograms for Compensation When performing a multi color experiment a non stained sample is run followed by the first single color stain to be analyzed e g FITC Create a series of plots such that this parameter is paired with each of the other colors in the experiment For example this 5 color experiment involves FITC PE PE
61. D Hodge and H A Young 2001 Activating Ly 49 NK receptors central role in cytokine and chemokine production J Immunol 166 8 4994 4999 Ortaldo J R A T Mason R Winkler Pickett A Raziuddin W J Murphy and L H Mason 1999 Ly 49 receptor expression and functional analysis in multiple mouse strains J Leukocyte Biol 66 3 512 520 Ortaldo J R R Winkler Pickett and G Wiegand 2000 Activating Ly 49D NK receptors expression and function in relation to ontogeny and Ly 49 inhibitor receptors J Leukocyte Biol 68 5 748 756 Ortaldo J R R Winkler Pickett J Willette Brown R L Wange S K Anderson G J Palumbo L H Mason and D W McVicar 1999 Structure function relationship of activating Ly 49D and inhibitory Ly 49G2 NK receptors J mmunol 163 5269 5277 Schaniel C L Bruno F Melchers and A G Rolink 2002 Multiple hematopoietic cell lineages develop in vivo from transplanted Pax5 deficient pre B I cell clones Blood 99 2 472 478 Voehringer D M Koschella and H Pircher 2002 Lack of proliferative capacity of human effector and memory T cells expressing killer cell lectinlike receptor G1 KLRG1 Blood 100 3698 Parasites Ellis T N and B L Beaman 2002 Murine polymorphonuclear neutrophils produce interferon y in response to pulmonary infection with Nocardia asteroides J Leukocyte Biol 72 373 Gao W H H Wortis and M A Pereira 2002 The Trypanosoma cruzi trans sialidase is a T cell ind
62. ES G1 R1 2 color demo fcs Iof x Uncompensated FITC Beads D GG 102 FITC Comp To properly set compensation adjust the X and Y axis sliders such that the median fluorescent channel of the positive population is nearly equivalent to the median fluorescent channel of the unstained population For example to set compensation for the FITC bead population align the FITC bead population within Region 5 so that the median fluorescent channel is nearly equal to CyAn ADP User Guide 65 the Region 4 population Within 1 0 is typically acceptable but more accuracy is usually obtained when performing compensation post acquisition I G1 R1 2 color demo fes OO x 101 102 103 FITC Comp 9842 7 23 5 05 2966 3 28 604 30 71 12 86 220 67 2340 3 28 2 21 lt 4465 626 43 zl 66 CyAn ADP User Guide Gi DakoCytomation To set compensation for the PE bead population align the PE bead population within Region 2 so that the median fluorescent channel is nearly equal to the Region 4 population EN G1 R1 2 color demo fes OF x 100 101 102 103 10t FITC Comp 7 23 5 05 2966 3 28 604 30 12 86 220 67 3 28 2 21 4465 626 43 2 29 2340 Advanced Issues This topic discusses the basic principle of spectral overlap between fluorescence detectors which creates a need for compensation An example of spectral overlap both in theory and in practice is shown for FITC and PE A number of c
63. Event Count Logarithmic Probability Equal Area High Density above Average Average Below Area Below Above Low Density Average Average 94 CyAn ADP User Guide dakocytomation Data Example The following is a example to illustrate the various contour methods A density plot displays the sample using the parameters FL1 versus FL2 Ei Contour Example 10 CyAn ADP User Guide 95 Using the same two parameters FL1 and FL2 the data is now displayed and compared using the four different contour methods Although data sets do vary the following is meant as a general guideline to illustrate how sample data is displayed using each contour method Notice how the linear method tends to highlight the peak data areas the log and equal area algorithms emphasize low density areas in comparison to the peak areas while equal probability provides the best overall representation of total events and their distribution B Contour Example E Contour Example 10 z 10 Log Density Bi Contour Example 104 104 Outlier Events Outliers refer to low density or low frequency populations When using various contour algorithms these populations can be lost or underrepresented in the dot plot The contour outlier 96 CyAn ADP User Guide vakocytomation option provides a mechanism to add dots or small lines in the plot to represent those events This option is useful when using either the Equal Probability or Linear
64. Gi DakoCytomation CyAn ADP User Guide Document Number 0000050 Revision B September 2004 Copyright 2004 DakoCytomation All rights reserved This document may not be copied in whole or in part or reproduced in any other media without the express written permission of DakoCytomation Please note that under copyright law copying includes translation into another language ii CyAn ADP User Guide dakocytomation The CyAn Advanced Digital Processing ADP High Performance Flow Cytometer is a research tool engineered for precision analysis of cells bacteria and other similarly sized particles With CyAn ADP DakoCytomation sets a new industry standard with a combination of features never before available on a bench top analyzer CyAn ADP gives users three excitation lines with independent alignment free focusing optics simultaneous 9 color and 2 scatter parameters analysis rates of 50 000 events per second a full 9 x 9 interlaser compensation matrix and high sensitivity The result is stable user friendly and flexible technology The instrument is optimized for cell cycle kinetics fluorescent protein work and multi color immunophenotyping Rare event analysis such as MHC Dextramers Tdex studies and no lyse whole blood applications are easily performed on the CyAn ADP The instrument also provides simplified compensation before during and after acquisition with unequaled sensitivity in all fluorescent channels
65. Hedlund S J Meech S Li J Schaack S P Hunger R C Duke and J DeGregori 1998 Adenoviral mediated gene transfer in lymphocytes PNAS 95 13159 Lewin S R R M Ribeiro G R Kaufmann D Smith J Zaunders M Law A Solomon P U Cameron D Cooper and A S Perelson 2002 Dynamics of T cells and TCR excision circles differ after treatment of acute and chronic HIV infection J Immunol 169 4657 146 CyAn ADP User Guide Gi DakoCytomation Li Y L Li R Wadley S W Reddel J C Qi C Archis A Collins E Clark M Cooley S Kouts H M Naif M Alali A Cunningham G W Wong R L Stevens and S A Krilis 2001 Mast cells basophils in the peripheral blood of allergic individuals who are HIV 1 susceptible due to their surface expression of CD4 and the chemokine receptors CCR3 CCR5 and CXCR4 Blood 97 3484 Li T and J Zhang 2002 Intramolecular recombinations of Moloney murine leukemia virus occur during minus strand DNA synthesis J Virol 76 9614 Mohri H A S Perelson K Tung R M Ribeiro B Ramratnam M Markowitz R Kost A Hurley L Weinberger D Cesar M K Hellerstein and D D Ho 2001 Increased turnover of T lymphocytes in HIV 1 infection and its reduction by antiretroviral therapy J Exp Med 194 1277 Okubo E J M Lehman and T D Friedrich 2003 Negative regulation of mitotic promoting factor by the checkpoint kinase Chk1 in simian virus 40 lytic infection J Virol 77 125
66. J A and I W Dawes 1998 Hydrogen peroxide causes RAD9 dependent cell cycle arrest in G2 in S cerevisiae whereas Mendadione causes G1 arrest independent of RAD9 function J Biol Chem 273 15 8564 8571 Zheng B J N Wu W Schober D E Lewis and T Vida 1998 Isolation of yeast mutants defective for localization of vacuolar vital dyes PNAS 95 11721 CyAn ADP User Guide 147
67. N Mavaddat K Winkel and K Shortman 2001 Human thymus contains 2 distinct dendritic cell populations Blood 97 1733 Villadangos J A M Cardoso R J Steptoe D van Berkel J Pooley F R Carbone and K Shortman 2001 MHC class II expression is regulated in dendritic cells independently of invariant chain degradation Immunity 14 739 Vremec D J Pooley H Hochrein L Wu and K Shortman 2000 CD4 and CD8 expression by dendritic cell subtypes in mouse thymus and spleen Journal of Immunology 164 2978 2986 Wang Y C G Kelly J T Karttunen T Whittall P J Lehner L Duncan P MacAry J S Younson M Singh W Oehlmann G Cheng L Bergmeier and T Lehner 2001 CD40 is a cellular receptor mediating mycobacterial heat shock protein 70 stimulation of CC chemokines Immunity 15 971 Wu L A D Amico K D Winkel M Suter D Lo and K Shortman 1998 RelB is essential for the development of myeloid related CD8a dendritic cells but not of lymphoid related CD8a dendritic cells Immunity 9 839 Yang O O F K Racke P T Nguyen R Gauslking M E Severino H F Horton M C Byrne J L Stroninger and S B Wilson 2000 CD1d on myeloid dendritic cells stimulates cytokine secretion from and cytolytic activity of Va24JaQ T cells A feedback mechanism for immune regulation J Immunol 165 3756 Drosophila Bryant Z L Subrahmanyan M Tworoger L LaTray C R Liu M J Li G van den Engh and H Ruohola
68. P User Guide Gi DakoCytomation How does this work with Dot Plots For example when setting compensation using FITC and PE beads or for a 2 color experiment unstained events that are FITC and PE would appear within the lower left portion of the FITC versus PE dot plot A double negative population A double positive population would appear somewhere within the upper right hand portion of the dot plot indicating both FITC and PE signal This population may appear along any fluorescent intensity range within the histogram based on factors such as FITC and PE fluorochrome density filters used or PMT voltage settings o jf CyAn ADP User Guide 59 In an ideal world where no compensation would be needed both the FITC and PE positive populations would fall along the appropriate axis and have the same median fluorescence as the negative population An ideal double negative and An ideal double negative and FITC positive population PE positive population oi e J The positive populations may appear anywhere along the corresponding axis The range of florescence intensity is based on factors such as antigen density the quantum fluorochrome excitation and emission efficiency filter differences or PMT voltage settings An ideal FITC positive population shown at varying degrees of signal intensity FITC _ FITC FITC i 60 CyAn ADP User Guide Ge DakoCytomation In reality because there is spectral
69. PI E3 f4 G6 R24 amp R1 amp R8 amp R2 All Stains PI EJ Ge R24 amp R1 amp R3 amp R2 All Stains PI E3 PE C y5 Comp 103 FITC Corre Non T or B cells Le NK cells CAS PR Cy5 cD3 FITO CDI FE CyAn ADP User Guide ap D 1P 40t PE Comp Non T or R cells i e NK cells Nan T or B cells i e NK cells CD45 PE Cy5 cbD45 CD3 FITS CD19 PE CD19 91 Contour Plots Contouring is a alternate method of data display available for 2 parameter histograms or plots When applied this option displays the data as a series of lines similar to what is observed with topographical maps based on the distribution and density levels of events in the plot Contour patterns will correlate to the density plot display but more importantly they can provide further insight into the distribution and number of events at various locations in the dot plot This property of contouring is useful for data analysis or for publication purposes Ei Contour Example 10 In Summit there are four contour algorithms available for displaying data Each of these methods contains a number of options where the assignment and mapping of lines can be adjusted As contour line patterns are modified the raw data values of the sample are not changed thus contouring only represents an alternative way to view and analyze the data Below is a listing of the available contour methods in Summit and a description o
70. TR Particles 1mL vial K0121 SpectraComp PE Cy5 Particles 1mL vial K0124 SpectraComp PE Cy7 Particles 1mL vial K0123 SpectraComp APC Particles 1mL vial K0124 SpectraComp APC Cy7 Particles 1mL vial CyAn ADP User Guide 123 Table A 2 Decontamination Clean Rinse Solutions Code Product 2323 Solution Clean amp Rinse 5 Liter 2324 Solution Decontamination 5 Liter To order CyAn ADP consumables contact DakoCytomation Sales at 1 800 822 9902 124 CyAn ADP User Guide 0 APPENDIX B TECHNICAL AND INSTRUMENT SPECIFICATIONS The technical and instrument specifications for the CyAn ADP are summarized in the following tables Table B 1 CyAn ADP UV Model Technical Specifications Performance Acquisition rate Up to 50 000 events sec Excitation Optics Optical parameters 2 scatter and 9 fluorescence Beam geometry Elliptical Spherical Number of excitation lines 3 Laser nominal operating output See Coherent 621 Operator s Manual for more information 488 nm 150mW argon UV 50mW argon 635 nm 25mW semiconductor Laser maximum output value Accessible in the interior of instrument 488nm 2W 351nm 6OmW 635nm 27 5mW Detectors and Filters User selected 488 nm Excitation FL1 530 40 nm FL2 575 25 nm FL3 613 20 nm FL4 680 30 nm FL5 750 nm UV Excitation FL6 400 40 nm FL7 450 50 n
71. acute lymphoblastic leukemia Blood 97 7 2115 2120 Coleman A B J Momand and S E Kane 2000 Basic fibroblast growth factor sensitizes NIH 3T3 cells to apoptosis induced by Cisplatin Molecular Pharmacology 57 324 333 Eischen C J M F Roussel S J Korsmeyer and J L Cleveland 2001 Bax loss impairs Myc induced apoptosis and circumvents the selection of p53 mutations during Myc mediated lymphomagenesis Molecular and Cellular Biology 21 22 7653 7662 Ghia P P Transidico J P Veiga C Schaniel F Sallusto K Matsushima S e Sallan A G Rolink A Mantovani L M Nadler and A A Cardoso 2001 Chemoattractants MDC and TARC are secreted by malignant B cell precursors following CD40 ligation and support the migration of leukemia specific T cells Blood 98 3 533 540 Girardi M D E Oppenheim C R Steele J M Lewis E Glusac R Filler P Hobby B Sutton R E Tigelaar and A C Hayday 2001 Regulation of cutaneous malignancy by y6 T cells Science 294 5542 605 609 Holash J Maisonpierre P D D Compton P Boland C R Alexander D Zagzag G D Yancopoulos and S J Wiegand 1999 Vessel cooption regression and growth in tumors mediated by angiopoietins and VEGF Science 284 1994 1998 Holtz M S M L Slovak F Zhang C L Sawyers S J Forman and R Bhatia 2002 Imatinib mesylate STI571 inhibits growth of primitive malignant progenitors in chronic myelogenous leukemia through reversal of abnormall
72. ade names and trademarks are the property of their respective holders CyAn ADP User Guide v vi CyAn ADP User Guide OR Table of Contents Heer RESOUICES seid s t DE tedden el dele ail Ae v ee v RI E UE v AAG SIMALKS isa crana gen GE ae v Tabie of Contents sersan a a aa aaa aa aa aen vii el r casssnacessacdieadeteciis 1 EE A E E E A nid ae ein ea nee ee ae 1 LASCr SACL E E eit eet ett eee ett nee eis 2 SECTION E A E a so auuadbenengesstsantaqebevenjacnensdaccenaneecevonunees 5 LAS CAM le RE 5 CyAn ADP Installation HReouirements ener ee ee tenets ee eeeeeeeaeeeeeeiaeeeeeenaeeeeneaes 5 General Laboratory Irtommaton cece eetteee erent eee ee tiene ee enieeeeeteeeeetnieeeeeeneeeeee 5 CyAn ADP Installation Requirements UV Model 6 General Laboratory Irtommaton erent eee eetieee ee ecieeeeetnneeeeesiieeeeeeneeeee 6 CyAn ADP UV Model Coherent Enterprise Laser Power Heouirements 7 CyAn ADP UV Model Coherent Enterprise Laser Ambient Air and Cooling Water SPOCIICALIONS secies i EEA EA E A AEEA AA E E EA AAEE dacaancesanavents 8 CyAn ADP UV Model Heat Exchange 8 ELE ET 9 System OVEN VIOW acras anaa a O TOT OOE EEEE 9 CYAN ARTE cbsernonsceetemeatretvonddepancsstansiae aivaieds EEN AOAO OET 9 CYAN ADP He Eu 10 IIIe e 11 Le 12 Electroni CS geduet edd eeEe deeg gedd Gg ie E ERNE AE EEEE A 16 Peripheral DEVICES i maticianciaccsandenaccaeantunstensats caadhatsdnwninda catceaudasae chad saa EE EES 16 SOM WAC ergeet cess A
73. al regulations Place a new sheath cubitainer back in its position on the cart Reconnect the cubitainer by using the quick connect If you are using the plastic carboy release the quick connects by pulling up on the collar Remove the carboy from the cart loosen the lid and fill with particulate free deionized water dH20 or a suitable sheath fluid Seal the lid and place the container back in its position on the cart Reconnect the sheath container using the quick connects Click Startup on the Cyan Control Panel Run SpectrAlign beads in accordance with your laboratory QC procedure Replacing Cleaner Fluid 34 Release the cleaner cubitainer quick connect Unscrew the cap from the spent cleaner cubitainer Retain this cap for use on the new cubitainer Dispose of the entire cleaner cubitainer in accordance with local state and federal regulations Remove the cardboard punch out on a new cleaner cubitainer Holding onto the ring around the cap pull up on the lid so that the lid extends up to the cardboard Remove the cap and replace it with the cap from the previous cubitainer Place a new cleaner cubitainer back in its position on the cart Reconnect the cubitainer quick connect CyAn ADP User Guide bakocytoma SECTION 6 CYAN ADP MAINTENANCE Regular maintenance of the CyAn ADP instrument is recommended as described in this section In addition to performing preventive maintenance procedures we also r
74. amples If not click Debubble on the CyAn Control Panel Rerun the SpectrAlign beads If necessary repeat Debubble and then recheck SpectrAlign beads IMPORTANT If you have problems with the CyAn ADP or maintenance questions please contact your local DakoCytomation Technical Service Group CyAn ADP Approved Cleaners and Disinfectants DakoCytomation Clean and Rinse DakoCytomation Decontamination Solution 70 Ethanol in DI water 0 1 Triton X100 in DI water 0 5N NaOH Sodium Hydroxide in DI water 10 Solution of household bleach in DI water 24 CyAn ADP User Guide dakocytomation Shutdown Procedure DakoCytomation recommends that Clean and DI Water Clean are enabled for shutdown 1 On the Edit menu click Preferences 2 Expand the Instrument list item by clicking the sign 3 Click CyAn 4 Inthe Command Options list select Shutdown check the Clean and DI Water Clean check boxes and then click Save and Close Preferences Preferences Command Options eeng Shutdown k Boost Wat 1 second FCS File 1 0 Debubble Debubble Count 2 Histogram Backflush s eege Clean Debubble Wait 6 25 Sort Master Clean amp Rinse Clean Time f1 l i CyCLONE Batch Sort Di Water Clean minutes Backflush Time 10 2 Parameters IW Auto Backflush Safe Sample PSI 0 05 ile SN Auto Sample E r Control Pane 3 35 Sample Regulator Min 8000 H Timout ae Regulator Max 65534 H Wait Ready Time 20 al GE Sheath W
75. an cvvslewee def E A dye ieee duvseael E PA T 16 SUNI WE 18 IO CUOM A E 21 Startup and Shutdown Procedures sssssssssresnseestttttttntttstttttt tnte nnsttt nt tnnn ansatt ttnn nannan nenene 21 Startup Procedure sernam airea a ea raa aaa aa ESA A E ERDE 21 Required ReageniS eege et Red EER 21 CYAN ADP Stamup EE 21 CyAn ADP Approved Cleaners and Disimtectamts 24 Shutdown e CEET 25 Section S isse ernas ececnnadsaaesecuteneenassansvecucekiencansestaetd nensiecasgaceaedpedenacdsanboesnaseerreesieouaediveetecis 27 Fluids Management i sc ccieetisiicettinde aiir nan EEN E ERRE E EEE 27 Sheath Management System Indicators essessssssseernanssnnnseennannnnnaneennaanannnnntenndanannaaeennaanana 27 Changing Out Sheath and Waste Containers 0 ccccecceeeee cence ee eeneeeeeeneeeeeetieeeeetneeeereaa 33 Replacing Cleaner Pluidseicccrcencessssanensysidecznesindedanepetdeaviwdecevapehcdeyyuvecdaueyetlebyveyehedevyweceseneied ice 34 DOCUOMN 65 siecctecscacectesscccencsscceeseesteceedsscecsfedsseeteessccedeeeascececeasteceeessccecerassedtedsscadeecdtsceceeeasteceeatiexerdescee 35 CyAn ADP Maintenance oeii NE ENEA ees 35 Daily Preventive Maintenance wicii ccccaesvescedesenetcceveetccaaveviicaaeseyeteaausvveneanavbncanetavdneeeeabveneanedanees 35 Weekly Preventive Maintenance ccccccccececeeeeeeeeeeeeeeeeeeeeeeeeseeeaeeeseeeaeeeseeeaeeeeseeaeeesenanees 35 SMS Clean amp Rinse Cycle ices icevveuteveved oo AEE EATA EEA 35 Monthly Preven
76. and dot plots in Summit only the number of channels is greater 256 4096 channels for histograms and 64 1024 channels for dot plots When data is initially acquired and saved the maximum resolution is imposed and the channel value is determined by the resolution of the instrument s analog to digital converter ADC The FCS file is saved as 10 bit data 1024 channels maximum 12 bit data 4096 channels maximum etc After the data is saved Summit allows it to be viewed and analyzed at 100 CyAn ADP User Guide vakocytomation various resolutions but not exceeding the resolution at which the data was collected analog to digitally converted and saved For example during analysis data from a 12 bit data file can be binned and displayed within a histogram using 4096 1024 or 256 channels A 12 bit data file can be analyzed using 256 or 1024 channels but not 2048 or 4096 since the later two cases exceed the resolution of the collected data The following illustrates this concept with a 2 bit data set 4 channels and 1 bit data set 2 channels The 2 bit data can be displayed and analyzed within either 2 or 4 channels but the 1 bit data set is limited to 2 channels You cannot exceed the maximum resolution of the raw data by binning extrapolation Changing Data Resolution Post Acquisition lt GE d 1 Bit 2 Bit 2 Channels Channels The following shows the data resolution settings available for histograms within Summit
77. and of all DakoCytomation employees is of primary concern Proper decontamination procedures must be followed for returned parts WARNING Wear Personal Protective Equipment in accordance with your laboratory safety procedures when operating or maintaining this instrument Use of this instrument in a manner other than that specified in this manual may cause impairment of equipment and result in injury Use of controls or adjustments or performance of procedures other than those specified in this manual may result in hazardous radiation exposure This radiation will be in the UV or visible regions of the electromagnetic spectrum Do not attempt to defeat the interlocks or open covers or panels retained with screws Laser Safety The CyAn ADP instrument conforms to international regulations encompassing laser safety The CyAn ADP is a Class 1 laser CLASS 1 LASER PRODUCT device IEC EN 60825 1 A2 2001 This designation indicates no hazardous laser energy is accessible to the user during normal operation or during a failure mode Note Because the CyAn ADP has been designed to operate as a Class 1 Laser Device the instrument must be operated with all light containment tubes in place and all protective light seals intact Most laser components of the CyAn ADP flow cytometer are Class IIIB to Class IV As such these lasers have the potential to cause injury Certification of regulatory compliance of these lasers often requires significant in
78. ar 1999 Sequence requirements of the GPNG Bum of the Ecballium elaterium trypsin inhibitor Il explored by combinatorial library screening J Biol Chem 274 30 21037 21043 Marine Biology and Limnology Andreatta S M M Wallinger T Posch and R Psenner 2001 Detection of subgroups from flow cytometry measurements of heterotrophic bacterioplankton by image analysis Cytometry 44 3 218 225 Button D K and B R Robertson 2001 Determination of DNA content of aquatic bacteria by flow cytometry Appl Environ Microbiol 67 4 1636 1645 DuRand M D R E Green H M Sosik and R J Olson 2002 Diel variations in optical properties of Micromonas pusilla Prasinophyceae J Phycol 38 1132 Robertson B R D K Button et al 1998 Determination of the biomasses of small bacteria at low concentrations in a mixture of species with forward light scatter measurements by flow cytometry Appl Environ Microbiol 64 10 3900 3909 Worden A Z S W Chisholm and B J Binder 2000 In situ hybridization of Prochlorococcus and Synechococcus with rRNA targeted peptide nucleic acid probes Appl Environ Microbiol 66 1 284 Monocytes Deszo E L D K Brake K A Cengel K W Kelley and G C Freund 2001 CD45 negatively regulates monocytic cell differentiation by inhibiting phorbol 12 myristate 13 acetate dependent activation and tyrosing phosphorylation of protein kinase Ca J Biol Chem 276 13 10212 10217 Eue l B Pietz J Storck
79. aste Pulse Width C Cube C Cube 05 H Min microsec Reusable Reusable 16 Max microsec EZ 25 6 i seconds seconds Laser 2 Delay Laser 3 Delay 5 2 e N Commit Changes H Save and Close 5 Click Shutdown on the CyAn Control Panel When prompted place a tube full of cleaner on the sample probe and close the lever Note Clicking Shutdown will automatically shut down the lasers and close the laser shutters If your CyAn is equipped with a remote control for the 488nm UV laser press the Off button 6 When prompted remove the tube replace with a tube of DI water and move the sample lever in 7 Check the status of the sheath container and the waste container by viewing the Sheath and Waste levels in the CyAn Control Panel Make sure the sheath container is at least half full and the waste container is at least half empty If necessary fill the sheath container and empty the waste container as described in Section 5 Fluids Management 8 Fill the test tube with DI water place it on the sample probe and leave the lever out CyAn ADP User Guide 25 9 For CyAn ADP UV model users only Turn the laser power supply key to the Off position and then turn the power switch to the Off position If desired secure the laser key On the LP5 check that the Cool Down Cycle indicator is illuminated If the indicator for the Cool Down Cycle is not illuminated make sure that the main
80. ated Green which depends on compensated Yellow For our example above we eliminated recursion by stopping at the first level where we substituted all raw events However if we did not stop at the first level of recursion we would ever more closely better approximate the biological reality For example consider our example event 3 Observed Values Event Yellow Green Blue 1 0 0 0 2 10 3 0 S 3 10 3 4 0 3 10 5 10 6 10 6 13 13 3 al 3 13 13 8 13 16 13 We see that event 3 is positive for Green but should be negative for both Yellow and Blue However because of our one step computation of compensated from compensated parameters we end up subtracting 0 3 30 of the one step compensated Green which is 8 from our Yellow value of 3 leaving 0 54 instead of the expected 0 0 Remember that the one step compensated Green will subtract 0 3 3 Yellow and 0 3 3 Blue from the original Green value of 10 leaving 8 However what if we permit this recursion What if we allow some level of recursion to take place where we actually do calculate compensated Yellow from compensated Green from compensated Yellow from compensated Green and so on The first approach is to see if the recursive nature of the operation will approach some limit that we can discreetly determine Unfortunately this is not the case please contact DakoCytomation directly if you want to understand the math theory as to why this cannot be solved However fa
81. ation in hardware An Alternative to Negative Compensation In response to these criticisms it turns out that there is a mathematically equivalent operation to bring about the same result Recall that historically compensation is performed on the raw data because the relatively simple analog compensation done in hardware has only the raw signal to work with However software compensation allows more flexibility in that it can do any amount of analysis in determining the compensated value for any given event and because no loss of resolution exists when parts of the signal are lost as a result of hardware compensation If we accept the fact that compensation on the raw signal results in over compensation and may even justify a negative compensation correction that leaves the possibility that compensating against a non inflated value against a compensated parameter may actually be a better approximation of biological significance This means we may want to consider compensating against compensated parameters instead of compensating against raw parameters whose signals may be artificially inflated Consider our original compensation equations Yellow comp Yellow 0 3 Green 0 0 Blue Green comp Green 0 3 Yellow 0 3 Blue Blue comp Blue 0 0 Yellow 0 3 Green If we were to compensate against compensated values the equations become Yellow comp Yellow 0 3 Green comp 0 0 Blue comp
82. ble stranded DNA B cells Immunity 16 535 546 Smith K G C D M Tarlinton G M Doody M L Hibbs and D T Fearon 1998 Inhitibition of the B cell by CD22 A requirement for Lyn J Exp Med 187 5 807 811 Tatu C J Ye L W Arnold and S H Clarke 1999 Selection at multiple checkpoints focuses V412 B cell differentiation toward a single B 1 cell specificity J Exp Med 190 7 903 914 Torres R M and K Hafen 1999 A negative regulatory role for Ig a during B cell development Immunity 11 527 536 Wang Y H R P Stephan A Scheffold D Kunkel H Karasuyama A Radbruch and M D Cooper 2002 Differential surrogate light chain expression governs B cell differentiation Blood 99 2459 Xu Y S J E Beavitt K W Harder M L Hibbs and D M Tarlinton 2002 The activation and subsequent regulatory roles of Lyn and CD19 after B cell receptor ligation are independent J Immunol 169 6910 Cancer Burchert A S Wolfl M Schmidt C Brendel B Denecke D Cai L Odyvanova T Lahaye M C Muller T Berg H Gschaidmeier B Wittig R Hehlmann A Hochhaus and A Neubauer 2003 Interferon a but not the ABL kinase inhibitor imatinib STI571 induces expression of myeloblastin and a specific T cell response in chronic myeloid leukemia Blood 101 259 Chen J S E Coustan Smith T Suzuki GA Neale K Mihara C H Pui and D Campana 2001 Identification of novel markers for monitoring minimal residual disease in
83. c myeloid leukemia Blood 101 259 Chatterjee S W Li C A Wong G Fisher Adams D Lu M Guha J A Macer S J Forman and K K Wong Jr 1999 Transduction of primitive human marrow and cord blood derived hematopoietic progenitor cells with adeno associated virus vectors Blood 93 1882 Clarke S H and L W Arnold 1998 B 1 cell development evidence for an uncommitted immunoglobulin lg M B cell precursor in B 1 cell differentiation J Exp Med 187 1325 140 CyAn ADP User Guide Gi oCytomation Dahl A M C Klein P G Andres C A London M P Lodge R C Mulligan and A K Abbas 2000 Expression of Bcl XL restores cell survival but not proliferation and effector differentiation in CD28 deficient T lymphocytes J Exp Med 191 2031 Fleming H E and C J Paige 2001 Pre B cell receptor signaling mediates selective response to IL 7 at the pro B to pre B cell transition via an ERK MAP kinase dependent pathway Immunity 15 521 Ghia P P Transidico J P Veiga C Schaniel F Sallusto K Matsushima S E Sallan A G Rolink A Mantovani L M Nadler and A A Cardoso 2001 Chemoattractants MDC and TARC are secreted by malignant B cell precursors following CD40 ligation and support the migration of leukemia specific T cells Blood 98 533 Goan S R Junghahn M Wissler M Becker J Aumann U Just G Martiny Baron Fichtner and R Henschler 2000 Donor stromal cells from human blood en
84. checked On For UV model users Make sure the LP5 or in house cooling system is turned on before turning on the 488nm UV laser If your CyAn is equipped with a remote control for the 488nm UV laser press the On button Jh On 488nm Deene M On 405mm m Opened M On 635mm m Opened i Shutdown Backflush Vacuum D Debubble Fluidics oft Cleanvtinse Preset 159 4 8 Open the required laser shutters by clicking the Closed buttons 9 On the Instrument menu point to CyAn and then click Clean to run the clean cycle with a tube of DakoCytomation Clean and Rinse solution or your choice of cleaner from the approved cleaner list see CyAn ADP Approved Cleaners and Disinfectants below 10 Repeat the clean cycle using a tube of DI water 11 Allow a minimum of 30 minutes for the lasers to stabilize 12 Open your QC file protocol generated by your laboratory place a tube with SpectrAlign beads 10 concentration on the sample probe and acquire F2 at a low event rate 100 eps Verify that the data is within your daily QC specification DakoCytomation recommends the following CV specifications for instrument calibration Parameter Target CV FL1 3 0 FL2 3 0 FL3 3 5 FL4 4 5 FL5 6 5 FL6 6 0 UV model 3 0 FL7 6 0 UV model 3 0 FL8 6 0 FLO 6 0 CyAn ADP User Guide 23 13 If QC data is within specification you are ready to run s
85. d photons is permitted to convert to a digital signal a numerical representation of how much light in a given spectrum was seen for that event then we have succeeded in getting the real or observed value into the computer and even saved in the data file Then because the real signal is preserved the researcher can play what if scenarios to compensate the data in various ways on the raw data to generate what the researcher believes to be the true signal Advantages The observed signal is not lost Compensation can be re applied in the future with different values for what if scenarios or to re create the experiment or generate in different ways what the researcher believes to be the true signal The computation can be the ideal computation where more than one color can be considered in compensating another color No restrictions are imposed for what colors may be compensated against other colors No specialized hardware is required Disadvantages The compensation algorithm is difficult to implement properly for both log and linear data but it can be done As technology continues to advance on so many levels an additional tool has been developed to address the historical weaknesses for sorting based on software or hardware compensation Digital Signal Processing DSP Compensation CyAn ADP User Guide 41 Digital Signal Processing DSP Compensation If the real or observed signal is still converted to
86. dendritic cells and HLA tetramers Blood 98 505 Usherwood E J R J Hogan G Crowther S L Surman T L Hogg J D Altman and D L Woodland 1999 Functionally heterogeneous CD8 T cell memory is induced by Sendai virus infection of mice J Virol 73 7278 van Rij R P H Blaak J A Visser M Brouwer R Rientsma S Broersen A M de Roda Husman and H Schuitemaker 2000 Differential coreceptor expression allows for independent evolution of non syncytium inducing and syncytium inducing HIV 1 J Clin Invest 106 1039 van Rij R P J A Visser R M van Praag R Rientsma J M Prins J M Lange and H Schuitemaker 2002 Both R5 and X4 human immunodeficiency virus type 1 variants persist during prolonged therapy with five antiretroviral drugs J Virol 76 3054 Williams O M K W Hart E C Y Wang and C M Gelder 2002 Analysis of CD4 T cell responses to human papillomavirus HPV type 11 L1 in healthy adults reveals a high degree of responsiveness and cross reactivity with other HPV types J Virol 76 7418 Zhang L S R Lewin M Markowitz H H Lin E Skulsky R Karanicolas Y He X Jin S Tuttleton M Vesanen H Spiegel R Kost J van Lunzen H J Stellbrink S Wolinsky W Borkowsky P Palumbo L G Kostrikis and D D Ho 1999 Measuring recent thymic emigrants in blood of normal and HIV 1 infected individuals before and after effective therapy J Exp Med 190 725 Yeast Flattery O Brien
87. e However in this case the compensated values for both particles were the same Both were determined at channel 986 5 instead of their true channel 1000 a 1 35 shift but the population was not further distorted indeed the population was tighter since both ended up in the same channel Using the proper calculation we correctly reach FITC true FITC measured 0 05 PE true PE true PE measured 0 30 FITC true Proper Proper Proper Proper Particle FITC PE FlTCmeasure FiTCtrue FITCcomp Diffftrue comp PErmeasure PEtrue PEcomp Diffftrue comp 1 100 100 100 D 100 100 100 D 2 1000 1000 1000 D 370 100 100 D 3 145 100 100 a 1000 1000 1000 D 4 1045 1000 1000 D 1270 1000 1000 D As seen in this calculation the computed value resolved to the true value with no population spreading and no population shift 56 CyAn ADP User Guide G DakoCytomation 2 Color Compensation Experiment A 2 Color Experiment involving FITC and PE The requirement for compensation arises from spectral overlap between fluorescence detectors Dyes such as fluorescein FITC and phycoerythrin PE which are excited at 488 nanometers will fluoresce optimally at 520 and 576 nanometers respectively Filters with bandwidths of 20 or 30 nanometers are typically used to allow photodetectors to respond to either a FITC or PE fluorochrome However the emission wavelengths of these dyes are sufficiently broad that light from a PE fluorochr
88. e Cy5 PEmeasure Cy5 PEtrue 1 100 100 100 100 100 100 d 1000 1000 370 100 100 100 3 145 100 1000 1000 172 100 4 1045 1000 1270 1000 172 100 5 109 100 325 100 1000 1000 6 1009 1000 595 100 1000 1000 7 154 100 1225 1000 1072 1000 8 1054 1000 1495 1000 1072 1000 Calculating 3 Color Compensation Values Using the historical calculation of compensation where we subtract percentages from the measured values not the true values we incorrectly reach FITC true FITC measured 0 05 PE measured 0 01 Cy5 PE measured PE true PE measured 0 30 FITC measured 0 25 Cy5 PE measured Cy5 PE true Cy5 PE measured 0 00 FITC measured 0 80 PE measured CyAn ADP User Guide 69 Historical Historical Historical Historical Historical Historical Particle FITC PE Cy5 PEFITCmeasure FiTCtrue FiTCcomp Dififtrue comp PEmeasure PEtrue PEcomp Diff true comp Cy5 PEmeasure Cy5 PEtrue Cy5 PEcomp Diff true comp i A U 100 100 100 0 100 100 100 0 100 100 10 0 2 e 1000 1000 986 5 135 370 100 100 0 100 100 78 4 216 3 145 100 99 28 0 72 1000 1000 968 5 31 5 172 100 100 0 4 1045 1000 985 78 14 22 1270 1000 968 5 315 172 100 78 4 216 5 109 100 88 75 11 25 325 100 973 27 1000 1000 982 18 6 1009 1000 975 25 24 75 595 100 97 3 27 1000 1000 950 4 39 6 7 154 100 88 03 1 97 1225 1000 9658 34 2 1072 1000 982 18 8 1054 1000 974 53 25 47 1495 1000 955 8 3
89. e FITC versus PE plot A slight adjustment was also needed for the FITC versus PE Cy5 plot Notice that the median values between the two populations is now within 1 0 for all 4 plots _ G1 RIPFITC ap am FITC Comp roei 10000 5 7031 100 00 o om D 9 o 000 0 0w 0 Da0 2316 327 i 206 3271 4765 67 29 rae 4765 67 29 70m 10000 52 33 205 me 0 om 0 00 000 o ooo O00 0 00 o Ow 00 Q00 0 600 O00 0 00 2322 3279 2 74 205 222 3279 274 1 38 4759 6721 Do 205 459 67 21 69 78 1 38 CyAn ADP User Guide 77 Running the Second Sample Next run the second single color sample Before doing this change the parameters of each of the histograms such that the second color in this case PE is plotted against the other 4 colors PE vs FITC PE vs Cy5 PE vs APC and PE vs APC Cy7 As the sample is run two populations will be delineated PE negative and positive G1 R1 PE Piles al i G1 R1 PE SS T 2433 10000 4 2433 100 00 o of s D U 000 ooo 0 00 1 oo 47 1 93 37855 32 78 z164 88 2164 8894 39 1 91 Ag 222 912 271298 13 82 e 2433 100 00 D aw JI o goo o om 0 O00 ZIA 88S 2164 8894 269 11 06 2397 14 2 259 11 06 78 CyAn ADP User Guide Gi DakoCytomation Again compare the median fluorescence among the negative and positive PE populations and adjust compensation as needed Below compensation was adjusted for both the PE versus FITC and PE versus PE Cy5 plots LU G1 R1
90. e along the axis increments by 1 2 or is reduced or scaled down by 50 to illustrate the scaled up data Ei Data Scaling Example Ei Data Scaling Example 1500 500 1125 750 375 0 Set Fixed Scale The Set Fixed Scale option allows you to determine an exact count value for the Y axis Simply select the checkbox to activate and enter a value for the scale Manual Scale oe Initial Range 135 a Increment by E Set Fixed Scale 1500 4 Se Scale The histogram and corresponding data will be set to the defined count value 110 CyAn ADP User Guide dakocytomation Ei Data Scaling Example 1500 Ei Data Scaling Example 1123 842 Auto Scaling During acquisition and for offline data analysis this feature is automatically performed in all histograms Users have the option to manually scale data in any histogram but by default auto scaling is initially activated Auto scaling ensures that the display of data in a histogram based on count number is visually proportional relative the scale of the Y axis based on the distribution and count number of the data Auto scaling also provides a proper visual display of events during acquisition For example if acquisition were performed without auto scaling the Y axis count value would be arbitrarily set As a result collected data for the sample would be initially displayed and confined to the histogram s visual area However as more events are collected
91. e container and the sheath container from the cart before emptying or filling Dispose of the contents of the waste container in accordance with local state and federal regulations WARNING Do not drop the waste or sheath containers on the Sheath Management System Doing so may result in improper calibration of the load cells 2 At the waste container release the quick connects 3 Remove the waste container from the cart remove the lid and empty Dispose of the contents of the waste container in accordance with local state and federal regulations 4 Place 40mL of regular household bleach into the bottom of the container Note This will provide 100ppm available chlorine when the tanks full capacity of 20L has been reached If the samples you are running will not be effectively killed by this concentration of sodium hypochlorite solution a larger amount of bleach should be used to achieve effective disinfection concentration 5 Replace the lid and tighten place the container back in its position on the cart and reconnect the waste container using the quick connects CyAn ADP User Guide 33 Restore the sheath fluid by either replacing the entire sheath cubitainer or refilling the plastic carboy depending on which type of sheath container is used with your Sheath Management System If you are using the sheath cubitainer release the quick connects Dispose of the entire cubitainer in accordance with local state and feder
92. e interior of instrument 488nm 20mW 635nm 27 5mW 405 nm 27 5mW Detectors and Filters User selected 488 nm Excitation FL1 530 40 nm FL2 575 25 nm FL3 613 20 nm FL4 680 30 nm FL5 750 nm 405 nm Excitation FL6 450 50 nm FL7 530 40 635 nm Excitation FL8 665 20 nm FL9 750 LP Signal Processing Compensation 9 x 9 full matrix Signal resolution 65536 Summit displays up to 4096 channels on all parameters Data acquisition channels 11 Fluidics Fluidics control system Software hardware and smart sensor controls provide ease of use with RUN and PAUSE modes CyAn ADP User Guide 127 Sample flow rate Up to 300 L min Sheath fluid Maximum 1 03 bar 15 psi nominal 0 41 bar 6 psi Quartz cuvette UV grade fused silica with 250 um square sectioned internal channel Summit Workstation Platform Windows XP or NT Processor 1 7 GHz or faster Memory 1 GB RAM minimum Storage space 2 60 GB hard drives minimum CDRW Monitor 17 inch LCD flat screen dual monitor also available Network High speed Ethernet 10 100 MB sec 128 CyAn ADP User Guide OR Table B 3 CyAn ADP Instrument Specifications CyAn ADP Enclosure Type Molded RIM polyurethane structural foam Installation Indoor only Height UV Model Dimensions not including Utility Cart Heat Exchanger or S
93. e of ways to calculate positive populations The two available options Channel by Channel and Overton can be accessed by right clicking within the subtraction histogram and selecting Subtraction Method from the popup menu Subtraction FL 2 20 Show Legend 12 Subtraction Method Overton T 1 New Bar Region wi Channel by Channel 1C fase add Histogram Data Set Fixed Normalization wi Auto Show Statistics Properties The Channel by Channel method subtracts all the collected events of the control from the sample data on a bin by bin or channel by channel basis The event difference is then reported for all channels and represents the true positive population Again keep in mind that which data set is designated as the control and which is the sample will mathematically effect the reported difference in the two samples For example the following represents two subtraction data sets the control and sample for a given parameter Events recorded for both groups are plotted within 6 channels and the calculated positive output is seen below Control events are subtracted from sample events with any positive difference reported Negative values are simply reported as 116 CyAn ADP User Guide control Sample Positives If we take the same two data sets but reverse which is the control versus the sample the following difference is reported Again sample events minus the control events on a per channel basis Notice
94. ecommend that you establish and perform other laboratory procedures for routine operations such as backing up your data and experimental protocols Daily Preventive Maintenance Follow the decontamination and cleaning procedures in Section 4 for daily preventive maintenance Weekly Preventive Maintenance SMS Clean amp Rinse Cycle Note Before running the clean amp rinse cycle make sure you have enough sheath fluid and cleaner fluid to complete the cycle On the CyAn Control Panel click the Clean and Rinse button A message box will warn you that the Clean and Rinse process will take 10 15 minutes Click OK to continue The clean cycle takes seven minutes to complete and the rinse cycle takes seven minutes to complete Monthly Preventive Maintenance Laser Interlock Maintenance Procedure 1 Follow the instrument startup procedure Make sure the lasers are on and the laser shutters are open 2 Visually inspect the housing on the instrument to verify that panels or covers are fitted and tight so that all laser energy is contained in the interior 3 Facing the instrument lift the lid approximately one inch The LED light s on the right front of the cover should go out verifying that the laser shutters are closed 4 Close the lid 5 Ifthe LED light s did not go out when the lid was opened contact your local DakoCytomation Support Representative CyAn ADP User Guide 35 Annual Maintenance Procedure A DakoCytoma
95. ependent B cell mitogen and an inducer of non specific Ig secretion Int Immunol 14 299 Mayer W E T Uinuk ool H Tichy L A Gartland J Klein and M D Cooper 2002 Isolation and characterization of lymphocyte like cells from a lamprey PNAS 99 14350 Stem Cells Akashi K X He J Chen H lwasaki C Niu B Steenhard J Zhang J Haug and L Li 2003 Transcriptional accessibility for genes of multiple tissues and hematopoietic lineages is hierarchically controlled during early hematopoiesis Blood 101 383 Asakura A P Seale A Girgis Gabardo and M A Rudnicki 2002 Myogenic specification of side population cells in skeletal muscle J Cell Biol 159 123 Bannert N M Farzan D S Friend H Ochi K S Price J Sodroski and J A Boyce 2001 Human mast cell progenitors can be infected by macrophagetropic human immunodeficiency virus type 1 and retain virus with maturation in vitro J Virol 75 10808 Batten M J Groom T G Cachero F Qian P Schneider J Tschopp J L Browning and F Mackay 2000 BAFF mediates survival of peripheral immature B lymphocytes J Exp Med 192 1453 Burchert A S Wolfl M Schmidt C Brendel B Denecke D Cai L Odyvanova T Lahaye M C Muller T Berg H Gschaidmeier B Wittig R Hehlmann A Hochhaus and A Neubauer 2003 Interferon o but not the ABL kinase inhibitor imatinib STI571 induces expression of myeloblastin and a specific T cell response in chroni
96. eq uirements Power Coherent Enterprise Il 621 Laser 208 240VAC single phase 50A Cooling method Water Cooling load 4 5 kW 15 000 Btu hr Cooling water pressure 138 414 kPa 20 60psi Cooling water inlet temperature 10 60 C 50 140 F Cooling water flow rate 8 11 6 I min 2 3 gpm CyAn ADP UV Model Heat Exchanger Option Coherent LP5 Heat Exchanger Height 54 0cm 21 0 in Width 69 0cm Depth 44 0cm 17 0 in Weight 50 kg 110 Ibs 27 0 in Power Coherent LP5 Heat Exchanger 110 220 VAC 10 50 60 Hz 10A Fuse Coherent LP5 Heat Exchanger power Type 5x25mm fast acting high capacity IEC 115 VAC operation 5A 230 VAC operation 2 5A Cooling Method Water to Air Heat Exchange Heat Output 5 kW 17 000 Btu hr Cooling water flow rate 8 9 5 I min 2 2 5 gpm Maximum laser water return temperature 68 C 154 4 F Maximum ambient air temperature 40 C 100 F CyAn ADP User Guide 131 heat exchanger only 132 CyAn ADP User Guide Gi oCytomation APPENDIX C FLOW CYTOMETRY REFERENCES Researchers around the world and across many scientific disciplines rely on the MoFlo High Performance Cell Sorter and the CyAn ADP High Performance Flow Cytometer A partial list of peer reviewed journal articles highlighting some
97. erent values 9 1 and 8 2 respectively The similar problem exists with events 6 and 8 that shared an original observed value of 13 but after compensation have 9 1 and 8 2 respectively In fact the problem even occurs with Yellow negative event pairs like 1 and 4 and 3 and 7 where values even calculate to a negative number This of course would crash the events into the axis on a histogram plot a typical problem for compensated data The problem for compensating Yellow in this example is the spectral overlap from Blue to Green where Green is artificially high on Blue positive events Because Blue makes Green artificially CyAn ADP User Guide 49 high we re subtracting too much Green from our Yellow but only on the Blue positive events This is an unfortunate problem for hardware compensation leading to population spreading as classical compensation is introduced Blue positive and Blue negative events separate when considering Yellow even though there is theoretically no spectral overlap from Blue to Yellow This is the anomaly for analog subtraction compensation in hardware and why some not all of the data is actually over compensated If you perform this calculation and never save the raw event data your population ends up being distorted This stretching isn t real it s a mathematical anomaly As Stanford pointed out this can be corrected with a mathematical adjustment to correct after the fact for the mathemat
98. essel A number of electrically controlled valves control the flow state of the system and provide a means for cleaning and debubbling in addition to routine flow conditions for sample analysis Sample is forced through tubing is introduced to the flow cell and is fluidically focused by sheath fluid This hydrodynamic focusing effect causes individual particles to be introduced in single file to each of the sequential laser beams While a pinch valve is used to allow the flow of sample the rate of sample flow is controlled by adjusting the over pressure differential pressure of an electrically controlled air regulator relative to sheath pressure Waste is drawn by a vacuum pump to a holding container for disposal System Valve Lever Figure 3 2 CyAn ADP Fluidics Block Diagram CyAn ADP User Guide 11 Optics Figure 3 3 is an illustration of the optical geometry of the CyAn ADP UV model Up to three excitation sources can be accommodated on the optical bench Each path has its own independent unique steering and focusing elements to provide optimal excitation of particles at the interrogation point Shown in the figure are laser paths for a 488 nm line blue a UV line and a 635 nm line red Each laser beam is focused to the quartz flow cell where particles are transported past the laser beams Steering Tower d Enterprise 621 488nm amp UV Kee Detector Block Objective Lase
99. f the instrument but may require future service Sheath O Message 406 Sheath subsystem is halted Please check that you have sufficient sheath fluid Sheath 6 Message 407 Sheath subsystem switch error Service maybe required Sheath ofl Message 705 Waste subsystem is halted Please check waste tank level Waste Waste Message 730 Less than 30 min until the waste container is full Message 710 Less than 10 min until the waste container is full Please empty the waste container Waste SEN Message 700 Waste container is full Please empty the waste container Waste OD Pressure on SE pressure inside CyAn Check connection between F Vacuum a Low vacuum inside CyAn Check connection between CyAn ge CyAn ADP User Guide LO CyAn Control Panel Message Text O Press Message 997 Low sheath pressure in SMS D De wll Message 998 Waste subsystem halted due to loss of vacuum If during operation please check vacuum pump waste quick connect or waste tank level lt Message in top portion of Control Panel gt Message 999 SMS Power fault Reset SMS to continue If this problem reoccurs please call DakoCytomation for technical support Changing Out Sheath and Waste Containers Use the shutdown fluidics procedure to fill and empty sheath and waste containers when running samples 1 Click Fluidics Off on the CyAn Control Panel WARNING Remove the wast
100. f complexity depending on their needs The instrument control panel figure 3 6 provides access to laser control event rate settings system maintenance functions such as clean and rinse and Sheath Management System functions The sample parameters panel figure 3 7 has software controls for adjusting parameter settings such as threshold PMT voltage and gain User documentation for Summit is available from the Help menu 16 CyAn ADP User Guide dakocytomation IV On 488mm E Opened Sample Parameters E G IS Threshold o 4 Trigger Peak Area Log Voltage M On 405mm Opened H On 635nm Shutdown Backtlush Vacuum D Debuble Fluidics oft Clean tinse Sheath Management System Peak Area Log N A Peak drea Log 400 Peak Area Log 400 Peak drea Log 400 Peak drea Log 400 Peak drea Log 400 Peak drea Log 400 Peak drea Log 400 Peak Area Log 400 Peak drea Log 400 BPR RP bo BP BP bb bt OD a OC fo Oo fod Oo hoy Oo fay Oo DE Peak drea Log 400 Figure 3 7 Sample Parameters Panel Sheath Figure 3 6 Instrument Control Panel CyAn ADP User Guide 17 Summit Features Summit software offers the compensation flexibility intelligence organization and intuitiveness essential for today s advanced flow cytometry applications
101. f each 92 CyAn ADP User Guide G DakoCytomation Histogram Properties General Iw Enable Contours Smoothing Resolution Axis Contour Methods Linear 64 Axis Ticks Dot Plot Display Linear C 128 Bivariates Equal Area B Contours i Se Probability Data Resolution Levels Max Threshold 1 Max 100 Step Size 0 H 50 A Close Contour Methods Linear Contour lines are plotted as a function of the maximum event number Lines are equally spaced and are uniformly distributed based on the number of events per contour area This method emphasizes high density portions of the data while minimizing low density areas As such peak areas will typically contain high concentrations of contour lines whereas contour detail may be lost for low density populations Keep in mind that linear plots can be misleading when comparing overall count numbers between populations For example a dot plot may contain two populations with equal numbers of cells one population is defined as a tall tightly distributed peak and the second as a short broad peak In this case the number of contour lines for the first population will be more numerous thus giving the overall impression that the first population has a greater number of cells Log Contour lines are plotted as a function of the maximum event number but with the lines logarithmically spaced For example the first contour line C is set at a population s highest dens
102. ferative disease in mice by tyrosine kinase fusion oncogenes does not require granulocyte macrophage colony stimulating factor or interleukin 3 Blood 97 5 1435 Wulf G G R Y Wang Kuehnle D Weidner F Marini M K Brenner M Andreeff and M A Goodell 2001 A leukemic stem cell with intrinsic drug efflux capacity in acute myeloid leukemia Blood 98 4 1166 1173 Dendritic Cells Barchet W M Cella B Odermatt C Asselin Paturel M Colonna and U Kalinke 2002 Virus induced interferon c production by a dendritic cell subset in the absence of feedback signaling in vivo J Exp Med 195 507 Brawand P D R Fitzpatrick B W Greenfield K Brasel C R Maliszewski and T De Smedt 2002 Murine plasmacytoid pre dendritic cells generated from FIt3 ligand supplemented bone marrow cultures are immature APCs J Immunol 169 6711 Briken V R M Jackman G F M Watts R A Rogers and S A Porcelli 2000 Human CD1b and CD1c isoforms survey different intracellular compartments for the presentation of microbial lipid antigens J Exp Med 192 281 Edwards A D S P Manickasingham R Sporri S S Diebold O Schulz A Sher T Kaisho S Akira and C Reis e Sousa 2002 Microbial recognition via toll like receptor dependent and independent pathways determines the cytokine response of murine dendritic cell subsets to CD40 triggering J mmunol 169 3652 Fong L M Mengozzi N W Abbey B G Herndier and E G Engle
103. graft in NOD SCID mice Blood 96 3971 Gongora R R P Stephan Z Zhang and M D Cooper 2001 An essential role for Daxx in the inhibition of B lymphopoiesis by type interferons Immunity 14 727 Habibian H K S O Peters C C Hsieh J Wuu K Vergilis C Grimaldi J Reilly J E Carlson A E Frimberger F M Stewart and P J Quesenberry 1998 The fluctuating phenotype of the lymphohematopoietic stem cell with cell cycle transit J Exp Med 188 393 Henckaerts E H Geiger J C Langer P Rebollo G Van Zant and H W Snoeck 2002 Genetically determined variation in the number of phenotypically defined hematopoietic progenitor and stem cells and in their response to early acting cytokines Blood 99 3947 Herblot S P D Aplan and T Hoang 2002 Gradient of E2A activity in B cell development Mol Cell Biol 22 886 Izon D J J C Aster Y He A Weng F G Karnell V Patriub L Xu S Bakkour C Rodriguez D Allman and W S Pear 2002 Deltex1 redirects lymphoid progenitors to the B cell lineage by antagonizing Notch1 Immunity 16 231 Jackson K A T Mi and M A Goodell 1999 Hematopoietic potential of stem cells isolated from murine skeletal muscle PNAS U S A 96 14482 Jackson K A S M Majka H Wang J Pocius C J Hartley M W Majesky M L Entman L H Michael K K Hirschi and M A Goodell 2001 Regeneration of ischemic cardiac muscle and vascular endothelium by adult ste
104. he first and continue through all channels to be used in this experiment The following plot shows the proper settings for negative population in PE channel 268 T T 100 101 104 103 104 Fe CyAn ADP User Guide 45 Completing the Compensation Matrix Helpful Tips It is a good idea to be aware of the emission spectra of your fluorochromes before you set up compensation and run the experiment Consult a fluorochrome guide or reference to research the extent of spectral overlap The greater the overlap between two fluorochromes the greater the error will be in the analysis or sort 1 Start by placing the first fluorescence channel FITC data on the x axis and the second fluorescence channel compensated PE Comp on the y axis If compensation has not yet been applied to PE PE Comp will not appear Select PE and then compensate Left click the mouse pointer in the FITC column and the PE Comp row This will alter the value in the box so it can be adjusted Adjust compensation appropriately Plot each fluorochrome on the x axis and sequentially go through each of the other fluorochromes in the experiment along the y axis 2 Proceed by moving on to the next fluorochrome To do this you need to do two things right click on the y axis label and select PE TR Comp from the list and left click in the PE TR Comp row of the FL1 column in the Compensation Matrix There is no need to plot the same fluorochrome on both the x and y axis
105. hih C C M C Hu J Hu J Medeiros and S J Forman 1999 Long term ex vivo maintenance and expansion of transplantable human hematopoietic stem cells Blood 94 1623 Shih C C M C Hu J Hu Y Weng P J Yazaki J Medeiros and S J Forman 2000 A secreted and LIF mediated stromal cell derived activity that promotes ex vivo expansion of human hematopoietic stem cells Blood 95 1957 Shih C C Y Weng A Mamelak T LeBon M C Hu and S J Forman 2001 Identification of a candidate human neurohematopoietic stem cell population Blood 98 2412 Spyridonidis A M Schmidt W Bernhardt A Papadimitriou M Azemar W Wels B Groner and R Henschler 1998 Purging of mammary carcinoma cells during ex vivo culture of CD34 hematopoietic progenitor cells with recombinant immunotoxins Blood 91 1820 Stewart F M S Zhong J Wuu C Hsieh S K Nilsson and P J Quesenberry 1998 Lymphohematopoietic engraftment in minimally myeloablated hosts Blood 91 3681 Wright D E E P Bowman A J Wagers E C Butcher and I L Weissman 2002 Hematopoietic stem cells are uniquely selective in their migratory response to chemokines J Exp Med 195 1145 Wulf G G R Y Wang Kuehnle D Weidner F Marini M K Brenner M Andreeff and M A Goodell 2001 A leukemic stem cell with intrinsic drug efflux capacity in acute myeloid leukemia Blood 98 1166 Zhong J F Y Zhan W F Anderson and Y Zhao 2002 Mu
106. his minimizes user error and data misinterpretation Absolutely Perfect Compensation In this example we ve illustrated how we can perform traditional compensation or correct for mathematical anomalies with negative compensation or compensating from the compensated as opposed to compensating from the raw signal However is it possible to perform compensate perfectly to achieve perfect results regardless of other spectra that may be present whether it overlaps a given color or not Remember that in our example we started with negative values at 0 and 3 and positive values at 10 and 13 While our mathematical corrections in performing compensation eliminated population spread that s found in traditional compensation we still have the problem that we never get all negative events to exactly 0 and all positive events to exactly 10 That s what we would expect from a perfect compensation algorithm since that s exactly the biological reality There s actually a little more going on when we stated that negative compensation or compensating on compensated not raw signals are mathematically equivalent That s still true both approaches are mathematically equivalent other than the fact that the negative compensation compresses the scale a little bit The real problem relies in the recursive nature of compensating on compensated not raw where we stated that compensated Yellow depends on 52 CyAn ADP User Guide CB oCytomation compens
107. ibitor 9 is up regulated during accessory cell maturation and effector cell degranulation and its overexpression enhances CTL potency J Immunol 170 805 Ho I C D Lo and L H Glimcher 1998 c maf promotes T helper cell type 2 Th2 and attenuates Th1 differentiation by both interleukin 4 dependent and independent mechanisms J Exp Med 188 1859 Hori S M Haury A Coutinho and J Demengeot 2002 Specificity requirements for selection and effector functions of CD25 4 regulatory T cells in anti myelin basic protein T cell receptor transgenic mice PNAS 99 8213 Izon D J J A Punt L Xu F G Karnell D Allman P S Myung N J Boerth J C Pui G A Koretzky and W S Pear 2001 Notch1 regulates maturation of CD4 and CD8 thymocytes by modulating TCR signal strength Immunity 14 253 Kim J I C Ho M J Grusby and L H Glimcher 1999 The transcription factor c Maf controls the production of interleukin 4 but not other Th2 cytokines Immunity 10 745 Kita H S Matsumura X S He A A Ansari Z X Lian J Van de Water R L Coppel M M Kaplan and M E Gershwin 2002 Quantitative and functional analysis of PDC E2 specific autoreactive cytotoxic T lymphocytes in primary biliary cirrhosis J Clin Invest 109 1231 Kochenderfer J N S Kobayashi E D Wieder C Su and J J Molldrem 2002 Loss of T lymphocyte clonal dominance in patients with myelodysplastic syndrome responsive to immunosuppressi
108. ical anomaly introduced as a result of the analog hardware compensation To resolve this problem negative compensation suggests putting back a little bit of Yellow based on the amount of Blue for that event that was accidentally subtracted This should give us a better approximation of the biological reality and should help to minimize the spreading in a Yellow compensated parameter for Blue positive and Blue negative events In other words add the Blue color to the equation using a negative compensation constant negative compensation Because we know that Blue overlaps Green by 30 in this example we know that Green is artificially high by 30 of Blue Of that 30 we know that there will be another 30 carry over to Yellow because we also stated that Green carries over 30 to Yellow This suggests the Blue should be added back at 0 3 0 3 which is 0 09 or 9 Performing negative compensation for Blue on these events produces the following result 1 Yellow comp 0 0 3 O Green 0 09 O Blue 0 0 2 Yellow comp 10 0 3 3 Green 0 09 O Blue 9 1 3 Yellow comp 3 0 3 10 Green 0 09 3 Blue 0 27 4 Yellow comp 0 0 3 3 Green 0 09 10 Blue 0 0 5 Yellow comp 10 0 3 6 Green 0 09 10 Blue 9 1 6 Yellow comp 13 0 3 13 Green 0 09 3 Blue 9 37 7 Yellow comp 3 0 3 13 Green 0 09 13 Blue 0 27 8 Yellow comp
109. ical filters and PMTs within the detection block assembly of the CyAn ADP UV Model and CyAn ADP respectively Dichroic filters are located at 45 to the direction of light propagation while emission filters are located at 90 to the light path Filter sticks are interchangeable thus allowing custom configurations to be implemented Please contact DakoCytomation to order dichroic filter sets 14 CyAn ADP User Guide dakocytomation 488 nm Laser ssc FL1 FL2 FL3 ao FL5 PE PE ssc FITC PE TxRed Cys PE Photomultiplier Tubes GFP PI PerCP Cy7 7AAD C f Ei CH ES he sfe zk d 8 e R F 95 45DLP 95DLP 40DLP 30DLP irror Dichroic Filters 730DLP 485DLP Ba 650DLP mirror S Se Emission Filters ch qe ch cn ES LI Filter configurations are 3 easily modified to S CAS Y accommodate different ea applications amp Scatter FL6 FL7 from Point U NUU 635 nm Laser 405 nm Laser Figure 3 5 CyAn ADP Detector Block and Optical Filter Layout CyAn ADP User Guide 15 Electronics CyAn ADP has an array of electronic components The PC workstation is used for instrument control status and data acquisition functions and communicates with the CyAn ADP instrument through a high speed serial link Housed within the instrument is a state of the art electronic chassis that provides a communication backplane bus with a number of available card slots to allow modular connection of key electronic control and sensing components
110. iling some limit optimization we can actually perform this calculation in software on each event until the incremental value of the next recursive iteration is insignificant In short this can be done For each event in this example the values for each event asymptotically approach the expected result 1 Yellow comp 0 0 3 O Green comp 0 0 O Blue comp 0 2 Yellow comp 10 0 3 3 Green comp 0 0 O Blue comp 10 3 Yellow comp 3 0 3 10 Green comp 0 0 3 Blue comp 0 4 Yellow comp 0 0 3 3 Green comp 0 0 10 Blue comp 0 5 Yellow comp 10 0 3 6 Green comp 0 0 10 Blue comp 10 6 Yellow comp 13 0 3 13 Green comp 0 0 3 Blue comp 10 7 Yellow comp 3 0 3 13 Green comp 0 0 13 Blue comp Oe 8 Yellow comp 13 0 3 16 Green comp 0 0 13 Blue comp Is this realistic Yes Given today s hardware this computation is realistic on every event even in very large files CyAn ADP User Guide 53 2 Color Compensation Fluorescent compensation is the process by which the cross contamination error from spectral emissions from other than the marker of interest is mathematically removed In other words it is the process by which the true value of each marker is computed from the measured value of each marker by removing the contamination from other markers This is illustrated in the followi
111. ilter a small percentage of the total collected PE signal will be from FITC fluorescence the contribution of area d to the PE measure Consequently we must compensate the observed PE signal by subtracting out a percentage of the observed FITC signal The same is true for our need to compensate the observed FITC signal by subtracting out a percentage of the observed PE signal we subtract out the contribution of area b to the FITC measure This is explained in the following equations If multiple colors are being measured simultaneously there can be a cascading need for compensation across several measured fluorescent values This can be accomplished by creating a Compensation Matrix in the Summit Software program to mathematically correct for the multiple combinations of fluorescent overlap Goal of Compensation The goal of compensation is to apply the proper correction factor to each parameter so that the median fluorescence in the stained population is equivalent to the median fluorescence in the unstained population If a specimen is stained with only FITC then ideally we should not see any fluorescent intensity in the PE channel However this is not the case because of the overlap in emission around 575nm between the two fluorochromes Once the proper compensation value is applied the median fluorescent intensity for PE in a sample stained with only FITC is equivalent to the PE median in cells not stained with FITC 58 CyAn AD
112. in all compensation values as well as the uncompensated data for each parameter Flexibility Summit Software offers flexibility throughout the program to allow the data to be gathered and manipulated in ways that best suit the application and user For example virtually any logical expression can be defined using any number of regions in AND and NOT combinations for generating statistics including non rectangular regions with an almost limitless number of vertices In addition any and all parameters available with the CyAn ADP High Performance Flow Cytometer can be selectively stored including the ability to disable non contiguous groups of parameters Eliminating unnecessary parameters increases the display performance during acquisition and reduces data storage needs 18 CyAn ADP User Guide vakocytomation Intelligence While performing batch analysis Summit Software s intelligent parameter matching feature dynamically determines the correct parameter to display for a sample based on antibody stain name parameter type channel or a combination of these This feature works regardless of the type of instrument that generated the FCS listmode file for the sample Organization Summit s sample viewer tracks samples in user definable folders Tests and controls with different parameters can be grouped and samples can be dragged and dropped between folders to help organize information Statistics for each gate or region are
113. ipid Antigens J Exp Med 192 281 Buslepp J S E Kerry D Loftus J A Frelinger E Appella and E J Collins 2003 High Affinity Xenoreactive TCR MHC Interaction Recruits CD8 in Absence of Binding to MHC J Immunol 170 373 Cipriani B G Borsellino F Poccia R Placido D Tramonti S Bach L Battistini and C F Brosnan 2000 Activation of C C B chemokines in human peripheral blood y T cells by isopentenyl pyrophosphate and regulation by cytokines Blood 95 39 Cunard R D DiCampli D C Archer J L Stevenson M Ricote C K Glass and C J Kelly 2002 WY14 643 a PPARza ligand has profound effects on immune responses in vivo J Immunol 169 6806 Dahl A M C Klein P G Andres C A London M P Lodge R C Mulligan and A K Abbas 2000 Expression of Bcl XL restores cell survival but not proliferation and effector differentiation in CD28 deficient T lymphocytes J Exp Med 191 2031 Dorris D R and K Struhl 2000 Artificial recruitment of TFIID but not RNA polymerase II holoenzyme activates transcription in mammalian cells Mol Cell Biol 20 4350 Doyle A M A C Mullen A V Villarino A S Hutchins F A High H W Lee C B Thompson and S L Reiner 2001 Induction of cytotoxic T lymphocyte antigen 4 CTLA 4 restricts clonal expansion of helper T cells J Exp Med 194 893 Dyall R and J Nikolic Zugic 1999 The final maturation of at least some single positive CD4 hi thymocy
114. irst blush this appears to be a recursive calculation For example FITC true is calculated from PE true which is similarly calculated from FITC true If these circular dependencies exist can the value be calculated 70 CyAn ADP User Guide Gi oCytomation First the calculations could be made if each operation was theoretically taken to infinity and the results were propagated back to the highest level Similarly today s hardware could compute the value at one level of recursion and then a second level and if those levels are different to a significant digit the process could continue until a third level comparing with the second level This is a practical approach that would take only a handful of iterations and is quite reasonable on a per particle basis even real time during acquisition on today s hardware 5 Color Compensation Experiment The following 5 color experiment involves compensation and uses the following fluorochromes FITC PE PE Cy5 APC and APC Cy7 Whole blood is drawn from a donor and partitioned into 7 tubes Tube 1 is not stained with any fluorochrome a no stain control Tubes 2 6 are each stained using one of the 5 colors Cells within tube 7 are stained using all 5 fluorochromes Below is a table listing the 7 tubes fluorochromes used for each and the respective cell surface marker targeted Sample Target Stain Tube 1 None None Tube 2 CD3 FITC Tube 3 CD19 PE Tube 4 CD45 PE
115. is example the first channel Histogram scaling would then be based on the tall peak in channel 1 thereby making the populations of interest appear unusually small due to the low number of events in the population in comparison to the first channel The effect of the Ignore Lower Channels and Ignore Upper Channels feature when activated and when turned off can be seen in the two histograms below Ei Data Scaling Example 160 Ei Data Scaling Example 160 Lower or upper channels 120 determines scale Ma 120 can determine the scale Data area that Counts Counts CO CH 10t 10 FL CyAn ADP User Guide 113 Subtraction Histograms A subtraction histogram is an analysis tool that reports the fluorescent difference between a sample and a control tube for a particular parameter of interest The calculated difference is displayed within the histogram and represents a true percentage of positively labeled events or cells This population is mathematically determined by subtracting the control from the sample data Subtraction FL 2 1240 9304 Control 620 Positives Controls Versus Samples When working with subtraction histograms it is important to properly designate which data set is the control and which represents the sample Reversing these will result in the calculation of a different positive population For example the two subtraction histograms below contain the same two data sets
116. ity point The next contour line C2 is mapped at half height of the original or C 2 The next contour C3 is again mapped at half height of the second line or C2 2 Contour lines are plotted until a value below 1 X is reached This method is useful for enhancing low density areas of data while maintaining some emphasis on high event areas emphasis on low event populations is less than with the equal area algorithm One way to counter this method s reduced focus on low event populations is to use the outlier option This adds dots or small lines in the plot to represent those events that fall outside the lowest contour CyAn ADP User Guide 93 Equal Area Contour lines are calculated for the entire data set and are plotted such that an equal area space occurs between contour lines This method tends to emphasize low density sparsely populated portions of the data and while under representing high concentration areas of the data Although this method is useful for highlighting rare and low event populations when applied to most plots the data can be difficult to interpret In general this method should be used sparingly Equal Probability Contour lines are plotted as a percentage of the total events With this method a percentage can be defined which determines the percentage of a population that will fall outside the contour areas The area or space between contour lines can vary and is equivalent to the corresponding
117. ive compensation may be used to make up for inherent overcompensation resulting from hardware compensation You can refer to Stanford s published paper on 8 Color compensation which addresses the issue of negative compensation 8 Color 10 Parameter Flow Cytometry to Elucidate Complex Leukocyte Heterogeneity Mario Roederer Stephen De Rosa Rachel Gerstein Michael Anderson Marty Bigos Richard Stovel Thomas Nozaki David Parks Leonore Herzenberg and Leonard Herzenberg Department of Genetics Stanford University Palo Alto California Cytometry 29 328 339 1997 Why Negative Compensation On its surface it would seem illogical to have negative compensation Compensation is usually a mathematical manipulation to remove the influence of an overlapping color on a given parameter Thus negative compensation would be adding back a color to a parameter Why would you ever want to do this Stanford s initial work on this subject was performed with Beckton Dickinson FACScan units where compensation was provided at the hardware level and the analog signals were subtracted prior to conversion to a digital signal this was hardware compensation Further they were working with many overlapping colors the paper listed above mentions 8 colors While their paper suggested several things two points are key Fluorescent colors overlap more than you might first think the asymptotic tails extend quite far into other spectra Analog compe
118. lactamase enzyme fragments PNAS 99 3469 Wentzel A A Christmann T Adams and H Kolmar 2001 Display of passenger proteins on the surface of Escherichia coli K 12 by the enterohemorrhagic E coli intimin EaeA J Bacteriol 183 7273 Wentzel A A Christmann R Kratzner and H Kolmar 1999 Sequence requirements of the GPNG f turn of the Ecballium elaterium trypsin inhibitor Il explored by combinatorial library screening J Biol Chem 274 30 21037 21043 Worden A Z S W Chisholm and B J Binder 2000 In situ hybridization of Prochlorococcus and Synechococcus marine cyanobacteria spp with RRNA targeted peptide nucleic acid probes Appl Environ Microbiol 66 284 B Cell Allman D R C Lindsley W DeMuth K Rudd S A Shinton and R R Hardy 2001 Resolution of three nonproliferative immature splenic B cell subsets reveals multiple selection points during B cell maturation J mmunol 167 6834 6840 Ansel K M R B S Harris and J G Cyster 2002 CSCL13 is required for B1 cell homing natural antibody production and body cavity immunity Immunity 16 67 76 Arnold L W S K McCray C Tatu and S H Clarke 2000 Identification of a precursor to phosphatidyl choline specific B 1 cells suggesting that B 1 cells differentiate from splenic conventional B cell in vivo Cyclosporin a blocks differentiation to B 1 J Immunol 164 2924 2930 Batten M J Groom T G Cachero F Quian P Schneider J Tschopp J L Browning
119. lations including a four level gradient of error for the FITC negatives and a four level gradient of error for FITC positives We now repeat the calculation with the proper compensation formulae FITC true FITC measured 0 05 PE true 0 01 Cy5 PE true PE true PE measured 0 30 FITC true 0 25 Cy5 PE true Cy5 PE true Cy5 PE measured 0 00 FITC true 0 80 PE true Proper Proper Proper Proper Proper Proper Particle FITC PE Cy5 PEFITCmeasure FiTCtrue FiTCcomp Difiitrue comp PEmeasure PEtrue PEcomp Diff true comp Cy5 PEmeasure Cy5 PEtrue Cy5 PEcomp Diff true comp 1 100 100 100 0 100 100 100 0 100 100 100 0 2 1000 1000 1000 0 370 100 100 0 100 100 100 0 3 145 100 100 0 1000 1000 1000 0 72 100 100 0 4 1045 1000 1000 0 1270 1000 1000 0 172 100 100 0 5 109 100 100 d 325 100 100 0 1000 1000 1000 H 6 1009 1000 1000 0 595 100 100 0 1000 1000 1000 0 154 100 100 0 1225 1000 1000 0 1072 1000 1000 0 8 1054 1000 1000 0 1495 1000 1000 0 1072 1000 1000 0 As before we have no such mathematical anomalies with the proper compensation approach Proper Compensation Pragmatics Consider again the formulae for proper compensation in our three color example FITC true FITC measured 0 05 PE true 0 01 Cy5 PE true PE true PE measured 0 30 FITC true 0 25 Cy5 PE true Cy5 PE true Cy5 PE measured 0 00 FITC true 0 80 PE true At f
120. leaner fluid is low Please replace the cleaner solution and press the Startup button on the CyAn Control Panel Clean O O Message 302 Cleaner fluid is empty Please replace the cleaner solution and e press the Startup button on the CyAn Control Panel ean Message 304 Cleaner quick connect is not completely engaged Please 2 check your connection LS O Message 305 Internal reservoir overfilled This will not prevent operation of the instrument but may require future service Clean O O Message 306 Clean subsystem is halted Please check that you have sufficient cleaner fluid Clean e O Message 307 Cleaner subsystem switch error This will not prevent operation e of the instrument but may require future service ean an Message 430 Less than 30 min of sheath fluid is remaining Sheath O Message 410 Less than 10 minutes of sheath fluid is remaining Please T replenish your sheath fluid ea CyAn ADP User Guide 31 CyAn Control Panel Message Text O Message 401 Internal sheath reservoir level is low CyAn will stop soon Please replenish your sheath fluid Sheath O Message 402 Out of sheath fluid Replenish sheath and press the Startup Sheath button on the CyAn Control Panel ea A Message 404 Sheath quick connect is not completely engaged Please check 8 i your connection O Message 405 Internal reservoir overfilled This will not prevent operation o
121. lity of doublets increases when analyzing cells that tend to stick together or when flowing at high event rates or a high pressure differential Doublets Definition Doublets occur when two cells or particles cross the path of the laser beam so close in time that the resulting analog signal from the detector is not Gaussian Tip Proper sample preparation and pre filtering of the sample can minimize doublets or clumps of cells Beads can be sonicated to minimize doublets Doublet Handling Integrated Signal The Integrated Signal of a doublet will yield a greater value than a singlet On the contrary Peak height selection on the ADC would not provide any information that would distinguish between a singlet and a doublet Note Area signal processing is only available in Linear amplification Make sure the desired ADC is set to Area CyAn ADP User Guide 105 Pulse Width The pulse width is defined as the time it takes for the signal to go above and back below the threshold Doublets will stay above the threshold longer than a singlet and will therefore provide a measurement and a means of discriminating against doublets Integrated Signal Pulse Width 5 5 us deadtime Finding Doublets using Color Gating Color Gating is an excellent way to help find the doublet population in your histograms Use the color gating feature in Summit to define and help set up sorting regions for doublet discrimination 1 Define a
122. m 635 nm Excitation FL8 665 20 nm FL9 750 LP Signal Processing Compensation 9 x 9 full matrix Signal resolution 65536 Summit displays up to 4096 channels on all parameters CyAn ADP User Guide 125 Data acquisition channels 11 Fluidics Fluidics control system Software hardware and smart sensor controls provide ease of use with RUN and PAUSE modes Sample flow rate Up to 300 L min Sheath fluid Maximum 1 03 bar 15 psi nominal 0 41 bar 6 psi Quartz cuvette UV grade fused silica with 250 um square sectioned internal channel Summit Workstation Platform Windows XP Processor 1 7 GHz or faster Memory 1 GB RAM minimum Storage space 2 60 GB hard drives minimum CDRW Monitor 17 inch LCD flat screen dual monitor also available Network High speed Ethernet 10 100 MB sec 126 CyAn ADP User Guide Gi Table B 2 CyAn ADP Technical Specifications Performance Acquisition rate Up to 50 000 events sec Excitation Optics Optical parameters 2 scatter and 9 fluorescence Beam geometry Elliptical Number of excitation lines 3 Laser options nominal operating output See Coherent OPSL Operator s Manual for more information 488 nm 20 mW Semiconductor 635 nm 25 mW semiconductor 405 nm 25 mW semiconductor Laser maximum output value Accessible in th
123. m cells J Clin Invest 107 1395 Kaeser P S M A Klein P Schwarz and A Aguzzi 2001 Efficient lymphoreticular prion propagation requires PrP c in stromal and hematopoietic cells J Virol 75 7097 Kirby S W Walton and O Smithies 2000 Hematopoietic stem cells with controllable tEpoR transgenes have a competitive advantage in bone marrow transplantation Blood 95 3710 Kouro T K L Medina K Oritani and P W Kincade 2001 Characteristics of early murine B lymphocyte precursors and their direct sensitivity to negative regulators Blood 97 2708 Kouro T V Kumar and P W Kincade 2002 Relationships between early B and NK lineage lymphocyte precursors in bone marrow Blood 100 3672 Kubota H and L M Reid 2000 Clonogenic hepatoblasts common precursors for hepatocytic and biliary lineages are lacking classical major histocompatibility complex class antigen PNAS 97 12132 Kuehnle l M H Huls Z Liu M Semmelmann R A Krance M K Brenner C M Rooney and H E Heslop 2000 CD20 monoclonal antibody rituximab for therapy of Epstein Barr virus lymphoma after hematopoietic stem cell transplantation Blood 95 1502 Lu L S J Tung N Baumgarth O Herman M Gleimer and L A Herzenberg 2002 Identification of a germ line pro B cell subset that distinguishes the fetal neonatal from the adult B cell development pathway PNAS 99 3007 Majka S M K A Jackson K A Kienstra M W Majesky M
124. man 2002 Productive infection of plasmacytoid dendritic cells with human immunodeficiency virus type 1 is triggered by CD40 ligation J Virol 76 11033 Henri S J Curtis H Hochrein D Vremec K Shortman and E Handman 2002 Hierarchy of susceptibility of dendritic cell subsets to infection by Leishmania major Inverse relationship to interleukin 12 production Infect Immun 70 3874 Le Bon A G Schiavoni G D Agostino Gresser F Belardelli and D F Tough 2001 Type i interferons potently enhance humoral immunity and can promote isotype switching by stimulating dendritic cells in vivo Immunity 14 461 Montoya M G Schiavoni F Mattei Gresser F Belardelli P Borrow and D F Tough 2002 Type interferons produced by dendritic cells promote their phenotypic and functional activation Blood 99 3263 Moron G P Rueda I Casal and C Leclerc 2002 CD8a CD11b dendritic cells present exogenous virus like particles to CD8 T cells and subsequently express CD8a and CD205 molecules J Exp Med 195 1233 O Keeffe M H Hochrein D Vremec J Pooley R Evans S Woulfe and K Shortman 2002 Effects of administration of progenipoietin 1 Fit 3 ligand granulocyte colony stimulating factor and pegylated granulocyte macrophage colony stimulating factor on dendritic cell subsets in mice Blood 99 6 2122 2130 O Keeffe M H Hochrein D Vremec I Caminschi J L Miller E M Anders L Wu M H Lahoud S Hen
125. math involved In this case this phenomenon is amplified depending upon the amount of compensation applied based on the degree of overlap between the two signals CyAn ADP User Guide 63 Double negative and FITC positive Double negative and PE positive populations after compensation populations after compensation An Example Using Fluorescent Beads The following illustrates an example of bead data using FITC and PE stained beads The dot plot below shows three populations an unstained population a FITC positive population and a PE positive population Data was collected using DakoCytomation s FloComp beads 2 color demo fcs lolx Uncompensated PE Beads Uncompensated ar FITC Beads Compensating the Data Within the dot plot you will want to create a quad region The statistics median fluorescence generated from the quad region will be used to align the median fluorescence of the unstained 64 CyAn ADP User Guide vakocytomation and stained populations Actual placement of the quadrants depends on the data and on the scientist s preference but placement at one decade on a four decade resolution axis is common Using Summit you can right click along a dot plot axis and select the Adjust Compensation option When the adjust compensation feature is activated red slider arrows will appear along both the X and Y axis These sliders can be used to adjust compensation 2 G1 R1 2 color demo fes Pm
126. ment 71 Background METTEG 71 Setting Up Histograms for Compensation cceceeeceeeeeeeeeeeeeneeeeeeeneeeeeeneeeeeeteeeeeenaees 72 Running the First Samples ics cccecetecd ugeet pone ctendy eectleenttetaa NEEE teenie 76 Setting Be ele e RTE 77 Running the Second Germgple sgrin areren canes AANER EEN Ed 78 Running the Remaining Samples icenian a A EA 81 Running the All Stains Sample aseseesssssneesrnessnrnsssnnasnnrnnnnerneannnannniannasnannanaeanaannanane na 87 Putting Compensation Into Practice eensecst iiie iinis en ireann EREE ERAR 87 Pan Leucocytes TEE 88 Helper subset of the T cell subset of white cells 0 ececeescecceceeeeeeeeeeaeeeeeeeeeeeeesnnaeees 89 Suppressor subset of the T cell subset of white cells ccccecceeeceeeteeeeeeeeeeeeeeeteeteees 89 White cells that are neither T or B cells LeNkcells 90 COntTOUP PIOUS EE 92 Contour Methods eher gege eege See Seder Eege Gegen degen eegenen eege ener 93 SE 1AL1 eg S EE E eege E E ee EEN ee eege Eed 94 QUTIEFEVENIS eech eege e deeg dEr Deech asta atic ARC eege Aa AEN aA 96 Display Al LCE 97 Data jRESOIMON KE 98 The Analog Particle or Cell Gonal cccceeeeceeeeeeteneeeeeeeneeeeeeaeeeeeecaeeeesetieeeeeesiaeeeeeeaes 98 The Digital Signal and Software Data Display 99 Doublet Discrtminaton aa e a a aa a aa Ae aa ee aa TE E aa AEAT aAA ihah 105 Doublet Handling EE 105 Integrated Ke ET 105 CyAn ADP User Guide Pulse WW Lt BEE 106 Finding Do
127. mermann L Nunez R Rossman K Reid K Moelling G D Yancopoulos and D J Glass 1999 Differentiation stage specific inhibition of the Raf MIK ERK pathway by Akt Science 286 1738 1741 Roncarati R N Sestan M H Scheinfeld B E Berechid P A Lopez O Meucci J C McGlade P Rakic and L D Adamio 2002 The y secretase generated intracellular domain of B amyloid precursor protein binds Numb and inhibits Notch signaling PNAS 99 10 7102 7107 Rosen E D C Husi X Wang S Sakai M W Freeman F J Gonzalez and B M Spiegelman 2002 C EBPa induces adipogenesis through PPARYy a unified pathway Genes amp Development 16 22 26 Scheinfeld M H R Roncarati P Vito P A Lopez M Abdallah and L D Adamio 2002 Jun NH2 terminal kinase JNK interacting protein 1 JIP1 binds the cytoplasmic domain of the Alzheimer s B amyloid precursor protein APP J Biol Chem 277 5 3767 3775 Tomasson MH I R Williams S Li J Kutok D Cain S Gillessen G Dranoff R A Van Etten and D G Gilliland 2001 Induction of myeloproliferative disease in mice by tyrosine kinase fusion oncogenes does not require granulocyte macrophage colony stimulating factor or interleukin 3 Blood 97 5 1435 High Throughput Screening Ashcroft R G and P A Lopez 2000 Commercial high speed machines open new opportunities in high throughput flow cytometry Journal of Immunological Methods 243 13 Battye F L A Light and D M Tarlint
128. n ADP User Guide vakocytomation Ei Data Scaling Example Ei Data Scaling Example 160 2304 Hit Value qm 1724 1154 574 FL The Ignore Lower Channels and Ignore Upper Channels option is a useful aid for auto scaling Keep in mind when data is collected for a sample all events that occur in all channels are collected and saved with the FCS file However when acquiring data a percentage of lower and upper channels can be ignored when auto scaling This option simply disregards any events in those channels when determining the proper Y axis scale for displaying the data Again all data in those channels is still collected and saved with the FCS file Why ignore a certain of lower and upper channels when auto scaling Periodically when samples are run a number of events are seen to buildup in either the first or last few channels These events can be due to various factors such as sample debris dead cells or instrument noise By ignoring the counts in these channels auto scaling is set based on a maximum event count in a population s of interest The alternative is to have auto scaling based on the maximum event count in any channel However this method can present a problem for data display As the sample is run a number of counts could accumulate in the first or last few channels Scaling in the histogram would then be based on the channel containing the maximum event number for th
129. n Fc receptor related gene FcRX expressed in human and mouse B cells Int Immunol 14 1075 Fleming H E and C J Paige 2001 Pre B cell receptor signaling mediates selective response to IL 7 at the pro B to pre B cell transition via an ERK MAP kinase dependent pathway Immunity 15 521 531 Gartner F F W Alt R J Monroe and K J Seidl 2000 Antigen independent appearance of Recombination Activating Gene RAG positive bone marrow B cells in the spleens of immunized mice Journal of Experimental Medicine 192 12 1745 Glazier K S S B Hake H M Tobin A Chadburn E J Schattner and L K Denzin 2002 Germinal center B cells regulate their capability to present antigen by modulation of HLA DO J Exp Med 195 1063 Gongora R R P Stephen Z Zhang and M D Cooper 2001 An essential role for Daxx in the inhibition of B lymphopoiesis by type interferons Immunity 14 6 727 737 Hayden T A P Riegert and G H Kline 2002 Detection of Functional VH81X Heavy Chains in Adult Mice with an Assessment of Complementarity Determining Region 3 Diversity and Capacity to Form Pre B Cell Receptor J Immunol 169 1970 Herblot S P D Aplan and T Hoang 2002 Gradient of E2A activity in B cell development Molecular and Cellular Biology 22 3 886 900 Honczarenko M Y Le A M Glodek M Majka J J Campbell M Z Ratajczak and L E Silberstein 2002 CCR5 binding chemokines modulate CXCL12 SDF 1 induced resp
130. nfected with Epstein Barr virus express the restricted pattern of latent genes previously found only in Epstein Barr virus associated tumors PNAS CyAn ADP User Guide 145 97 12250 Bannert N M Farzan D S Friend H Ochi K S Price J Sodroski and J A Boyce 2001 Human Mast cell progenitors can be infected by macrophagetropic human immunodeficiency virus type 1 and retain virus with maturation in vitro J Virol 75 10808 Barchet W M Cella B Odermatt C Asselin Paturel M Colonna and U Kalinke 2002 Virus induced interferon a production by a dendritic cell subset in the absence of feedback signaling in vivo J Exp Med 195 507 Baric R S E Sullivan L Hensley B Yount and W Chen 1999 Persistent infection promotes cross species transmissibility of mouse hepatitis virus J Virol 73 638 Baron J L L Gardiner S Nishimura K Shinkai R Locksley and D Ganem 2002 Activation of a nonclassical NKT cell subset in a transgenic mouse model of hepatitis B virus infection Immunity 16 583 Baumgarth N O C Herman G C Jager L Brown and L A Herzenberg 1999 Innate and acquired humoral immunities to influenza virus are mediated by distinct arms of the immune system PNAS 96 2250 Baumgarth N O C Herman G C Jager L E Brown L A Herzenberg and J Chen 2000 B 1 and B 2 Cell derived Immunoglobulin M Antibodies Are Nonredundant Components of the Protective Response to Influenza Virus Infecti
131. ng graph Emission Spectra Wavelength nm Based on the labeled regions the true and measured values for each marker would be FITC true a FITC measured a b PE true c PE measured c d Calculating Compensation Values Historically fluorescent compensation to deduce the true from the measured for a two color experiment using FITC and PE was defined incorrectly as FITC true FITC measured C1 PE measured PE true PE measured C2 FITC measured The constants C1 and C2 are proposed to either be spillover coefficients based on the area percentage that PE fluorescence contributes to the measure in the FITC band above and the area percentage that FITC fluorescence contributes to the measure in the PE band above respectively or compensation coefficients based on the area percentage that other markers in addition to PE contributes to the measure in the FITC band above and the area percentage that other markers in addition to FITC contributes to the measure in the PE band above respectively Unfortunately this calculation is incorrect It is technically inappropriate and mathematically ambiguous to consider contamination from other colors as part of the computation for the true marker value Rather these equations should read FITC true FITC measured C1 PE true PE true PE measured C2 FITC true 54 CyAn ADP User Guide gt oCytomation The premise of the histo
132. nipulation against observed reality It can be considered falsification of data just as any arbitrary mathematical manipulation of results would be considered false It is improper to base research on anything other than the actual or observed reality This assertion is heightened by the fact that many users do not fully understand the issue of compensation how it is being performed and how compensated data is to be interpreted For example even though two populations can be mathematically be separated on the screen the overlap of the observed signal is the actual reality cell sort purities on a population identified by an experienced operator on raw data should be the same as cell sort purities on a population identified on compensated data no matter how good your compensation The argument for compensation Those who favor the use of compensation argue that it is a mathematical manipulation to bring the physical observation of reality in line with a biologically significant reality Thus compensation is merely a tool to assist the researcher in viewing the data in a format where biologically significant populations are more easily identified as opposed to relying on the researcher to perform the same conversion in his or her head CyAn ADP User Guide 39 This can be likened to the argument of estimating the next digit of precision when you can see a little further than the scale offers For example what is the width of the compone
133. nsation and software compensation Hardware Compensation The colors are first observed in analog because photons and the real world are in analog Before these signals or values are converted to digital the computer manipulates everything in digital format and event data is always stored in digital format colors can be subtracted from each other to bring the observed signal in line with what the researcher believes to be the true signal 40 CyAn ADP User Guide M dak Advantages The computation is very close to the ideal computation The data looks very good The electronics are relatively easy to implement Disadvantages The observed signal is usually lost Only the mathematically manipulated signal what the researcher believed at the time to be true is actually stored Because of this you can t re create your experiment from the data or change your mind later if your compensation constants were incorrect Because of hardware path limits you only have a fixed number of color paths that you are permitted to compensate For many hardware platforms this usually means you cannot compensate all your colors against each other even if you only have three colors The algorithm is still only approximate and the actual result is data distortion and over compensation subtracting too much This opens the topic of negative compensation Software Compensation If the analog signal of the observed light the observe
134. nsation at the hardware level is flawed in that it over compensates the data The first point suggests that the spectral overlap can be larger than the user first anticipates especially when considering the theoretical spectral curves for each color The second point suggests that additional colors other than those with expected spectral overlap may influence the compensation of a given color This influence results in overcompensation that must later be undone However if the overcompensation took place on analog data at the hardware level the original data is no longer available to correct for the initial overcompensation at the time the data was collected CyAn ADP User Guide 47 A Negative Compensation Example As a specific example consider the following event data where we have three colors Yellow Green and Blue Further consider that each color overlaps the neighboring color by 30 but that non neighboring colors do not overlap at all Note that in reality it is likely that non neighboring colors do actually overlap but that only complicates the math in this example the principle is the same Thus if you were to build a compensation matrix it would look like the following GG Compensation for Yellow Green and Blue KE Yelow Comp Yellow Comp Iw 30 00 0 00 Green Comp 30 00 Iw 30 00 Blue Comp 0 00 30 00 Iw We now generate a list of every possible combination for a particle with po
135. nt below One person might argue that your measurement is simply restricted to the resolution of your scale and you must pick 3 16 or 4 16 only In this case you d probably round down to 3 16 because the width appears to be closer to that mark than the next This same person would argue that you cannot make up new digits of precision because they simply do not exist at this scale However another individual may argue that you know it is in fact greater than 3 16 and less than 4 16 Further you can estimate the location in that range between the two marks In this case you are not actually making up new digits of resolution you simply take advantage of the resolution that is present in the measurement but not present in the current scale being used If the exact location were 4 16 of the way between the two marks and your guess was in fact 3 16 or 5 16 or even 2 16 or 6 16 between the two marks you would still be closer to the true measurement of the item and therefore this estimation of the next digit will actually increase the resolution of the measurement In terms of compensation the researcher is mathematically manipulating the data to better approximate the biological significance of the populations Where one scientist may consider it fabrication another may consider it enhanced resolution Hardware vs Software Compensation Historically there have been two approaches to performing compensation hardware compe
136. of their cutting edge science on the CyAn and MoFlo instruments follows Bacteria Button D K and B R Robertson 2001 Determination of DNA content of aquatic bacteria by flow cytometry Appl Environ Microbiol 67 1636 Christmann A K Walter A Wentzel R Kratzner and H Kolmar 1999 The cystine knot of a squash type protease inhibitor as a structural scaffold for E coli cell surface display of conformationally constrained peptides Protein Engineering 12 9 797 806 Edwards R A and S R Maloy 2001 Inside or outside Detecting the cellular location of bacterial pathogens BioTechniques 30 2 304 Gryllos l C Cywes MH Shearer M Cary R C Kennedy and M R Wessels 2001 Regulation of capsule gene expression by group A Streptococcus during pharyngeal colonization and invasive infection Molecular Microbiology 42 1 61 74 Robertson B R D K Button and A L Koch 1998 Determination of the biomasses of small bacteria at low concentrations in a mixture of species with forward light scatter measurements by flow cytometry App Environ Microbiol 64 3900 Sierig G C Cywes M R Wessels and C D Ashbaugh 2003 Cytotoxic effects of Streptolysin O and Streptolysin S enhance the virulence of poorly encapsulated Group A Streptococci Infect Immun 71 446 Wehrman T B Kleaveland J H Her R F Balint and H M Blau 2002 Protein protein interactions monitored in mammalian cells via complementation of B
137. of total events i e contour lines are closer together in high density areas Because the area between contour lines represents a fixed percentage of the total population an equal number of events will appear between each pair of contour lines The algorithm derives i s name from the fact that each event has an equal probability to fall between any pair of adjacent contours Overall this method provides a good representation of the total number of events in a population since in a given area the number of visible contour lines are fairly proportional to the number of events This is because events that fall between pairs of contour lines correspond to a certain percentage of events in the sample i e high concentration narrow lines low concentration wide lines Like linear density contouring this algorithm emphasizes high density areas However unlike the linear method equal probability provides a better portrayal of low event populations If necessary one way to increase the emphasis on low event populations is to use the outlier option This adds dots or small lines in the plot to represent those events that fall outside the lowest contour Summary The chart below presents a comparison between the different contouring algorithms Linear Log Equal Area and Equal Probability and how effective each is for emphasizing both high and low density events Below and Above refer to the under or over emphasis of a population type
138. om other spectrums that are not of interest and thus determine a rough FITC measure and a rough PE measure By rough we mean that this is only an approximate measure because some contamination remains from other fluorescence beyond the fluorescence from the single marker of interest For example let s again look at the FITC and PE emission spectra but also use filters and dichroics to isolate a frequency band to measure FITC and another frequency band to measure PE Emission Spectra Bandpass Filter 530 30 Bandpass Filter 585 42 vi TNE 502 552 2 652 Wavelength nm As shown in the graph the FITC measure will be based on the area under the FITC curve within the 530 30 band but will also include a small amount of contamination from the lower end of the PE curve that also extends into this 530 30 band Similarly the PE measure will be based on the area under the PE emission curve within the 585 42 band but with a relatively large amount of contamination from the upper end of the FITC emission curve that similarly extends into the 585 42 band The contamination of PE into FITC and FITC into PE is cross contamination and poses a potentially large basis for error in our measure This contamination in the measured range is termed spectral overlap 44 CyAn ADP User Guide G DakoCytomation Compensation Preparation Preparation Summary In order for compensation to be properly defined an n color experiment will
139. ome will leak through the filter of the FITC detector and vice versa Compensation is a method to remove this spectral overlap This makes the data orthogonal simplifying human interpretation and separating distinct populations that may overlap on the dot plot Emission Spectra for FITC and PE Fluorochromes a I S 5 o w a KI A e ie 5 z 580 Wavelength nm The diagram above shows the emission spectra for both FITC and PE It is common for a fluorochrome s emission distribution to have a steep intensity rise on the shorter wavelength side and a gradual decline in intensity on the longer wavelength side This usually means that spectral overlap is less a concern for the shorter wavelength fluorochrome than for the longer wavelength fluorochrome usually we worry about emissions from the shorter wavelength fluorochrome spilling into the bandpass filter range and PMT of the longer wavelength fluorochrome not the other way around In this case FITC contaminates PE more than PE contaminates FITC in the range we use to measure the fluorochrome CyAn ADP User Guide 57 Fluorescence Spillover between FITC and PE using Bandpass Filters PE 580 30 Bandpass FITC 530 40 Bandpass E KI z gt 580 Wavelength nm As seen when using a 580 30 bandpass filter to detect PE signal this spectral overlap of FITC within the filter s wavelength range causes a falsely high accounting of PE In the range of the bandpass f
140. ompensation The term compensation as applied to flow cytometry typically refers to a mathematical manipulation that subtracts or otherwise minimizes the effect of a spectral overlap across colors thereby better resolving sub populations More accurately compensation is the process by which the physical observation is mathematically manipulated to better observe biological significance A common problem in resolving events that are positive in one color from events in another color is that the spectrum may be very close together Worse yet the spectrums may actually overlap For example if you have a yellow dye and a green dye in the same sample it s possible that some events are only positive for yellow but they actually look a little green because yellow and green overlap a little bit in their spectrums As with any tool compensation is a not useful to a user that does not understand it While some would argue that properly performed compensation better identifies populations of biological significance almost all would agree that improper compensation is worse than no compensation It distorts data leads to inaccurate results and can even be considered fraudulent if done intentionally Still among very educated users that know the issue well there is little agreement over the acceptable use of compensation The argument against compensation Those who are against the use of compensation argue that it is a mathematical ma
141. ompensation topics exist which are well beyond the scope of this topic These include issues and differences in hardware versus software compensation online and post acquisition compensation different compensation algorithms and approaches mathematical anomalies that exist with some algorithms compensation for experiments with more fluorescent parameters five to seven parameters are common with some research at twelve or more simultaneous colors procedures to run single and multi color controls and procedural and operational considerations The latter includes various types of fluorochrome interaction competition background noise quantum fluorochrome excitation and emission efficiency filter differences PMT voltage settings etc While conceptually simple the need for proper fluorescent compensation plays a significant role in research advances involving instrumentation reagents and sample preparation or measure CyAn ADP User Guide 67 3 Color Compensation The issue of compensation is dramatically amplified with additional markers in the same experiment We now consider the following excitation curves for FITC PE and Cy5 PE a tandem dye where excitation is in a PE molecule but fluorescence is transferred to the Cy5 molecule for emission in a lower energy wavelength including the 488nm laser line used to excite each Excitation Spectra Wavelength nm FITC PE and Cy5 PE will fluoresce at the following wavelengths
142. on However after running the all stains sample how does one make sense of and use the compensated data from the all stains sample The following illustrates 4 different cell populations that can be identified analyzed and or sorted based on the 5 color experiment For each of the cell populations below identification is based upon the presence or absence of specific cell surface markers To recall which marker correlates to which fluorochrome refer to the original tube setup table below CyAn ADP User Guide 87 Sample Target Stain Tube 1 None None Tube 2 CD3 FITC Tube 3 CD19 PE Tube 4 CD45 PE Cy5 Tube 5 CD4 APC Tube 6 CD8 APC Cy7 Tube 7 AN Pan Leucocytes B cells Pan Leucocytes and B cells are both CD45 PE Cy5 and CD19 PE Using the all stains sample when a dot plot of compensated PE versus PE Cy5 is created as seen below the pan leucocytes and B cells appear in the upper left quadrant indicating a positive signal for both PE Cy5 and PE G1 R1 All Stains x 2 E 5 O We gt 9 w a Pan Leucocytes CD 45 PE Cy5 CD19 PE 88 CyAn ADP User Guide dakocytomation Helper subset of the T cell subset of white cells Helper cells a subset of the T cell population of white cells are CD45 PE Cy5 CD3 FITC and CD4 APC Using the all stains sample when 3 dot plots are created with compensated FITC versus PE Cy5
143. on 2000 Single cell sorting and cloning Journal of Immunol Methods 243 25 Brenner S M Johnson J Bridgham G Golda D H Lloyd D Johnson S Luo S McCurdy M Foy M Ewan R Roth D George S Elitr G Albrecht E Vermaas S R Williams K Moon T Burcham M Pallas R B DuBridge J Kirchner K Fearon J Mao and K Corcoran 2000 Gene expression analysis by massively parallel signature sequencing on microbead arrays Nature Biotechnology 18 630 634 Brenner S S R Williams E H Vermaas T Storck K Moon C McCollum J Mao S Luo J J Kirchner S Eletr R B DuBridge T Burcham and G Albrecht 2000 In vitro cloning of complex mixtures of DNA on microbeads Physical separation of differentially expressed cDNAs PNAS 97 4 1665 Christmann A K Walter A Wentzel R Kratzner and H Kolmar 1999 The cystine knot of a squash type protease inhibitor as a structural scaffold for E coli cell surface display of conformationally constrained peptides Protein Engineering 12 9 797 806 Daugherty P S B L lverson and G Georgiou 2000 Flow cytometric screening of cell based libraries Journal of Immunol Methods 243 211 Demo S D E Masuda A B Rossi B T Throndset A L Gerard E H Chan R J Armstrong B P Fox J B Lorens D G Payan R H Scheller and J M Fisher 1999 Quantitative measurement of mast cell degranulation using a novel flow cytometric annexin V binding assay Cytometry 36 340
144. on Blood 100 3639 Koehne G H F Gallardo M Sadelain and R J O Reilly 2000 Rapid selection of antigen specific T lymphocytes by retroviral transduction Blood 96 109 Koehne G K M Smith T L Ferguson R Y Williams G Heller E G Pamer B Dupont and R J O Reilly 2002 Quantitation selection and functional characterization of Epstein Barr virus specific and alloreactive T cells detected by intracellular interferon y production and growth of cytotoxic precursors Blood 99 1730 Lieberson R K A Mowen K D McBride V Leautaud X Zhang W K Suh L Wu and L H Glimcher 2001 Tumor necrosis factor receptor associated factor TRAF 2 represses the T helper cell type 2 response through interaction with NFAT interacting protein NIP45 J Exp Med 194 89 McGavin M K K Badour L A Hardy T J Kubiseski J Zhang and K A Siminovitch 2001 The intersectin 2 adaptor links Wiskott Aldrich Syndrome protein WASp mediated actin polymerization to T cell antigen receptor endocytosis J Exp Med 194 1777 Mohrs M K Shinkai K Mohrs and R M Locksley 2001 Analysis of type 2 immunity in vivo with a bicistronic IL 4 reporter Immunity 15 303 Monroe R J K J Seidl F Gaertner S Han F Chen J Sekiguchi J Wang R Ferrini L Davidson G Kelsoe and F W Alt 1999 RAG2 GFP knockin mice reveal novel aspects of RAG2 expression in primary and peripheral lymphoid tissues Immunity 11 201 Moron
145. on J Exp Med 192 271 Drexler l C Staib W Kastenmuller S Stevanovic B Schmidt F A Lemonnier H G Rammensee D H Busch H Bernhard V Erfle and G Sutter 2003 Identification of vaccinia virus epitope specific HLA A 0201 restricted T cells and comparative analysis of smallpox vaccines PNAS 100 217 Fong L M Mengozzi N W Abbey B G Herndier and E G Engleman 2002 Productive infection of plasmacytoid dendritic cells with human immunodeficiency virus type 1 is triggered by CD40 ligation J Virol 76 11033 Gao J B P De and A K Banerjee 1999 Human parainfluenza virus type 3 up regulates major histocompatibility complex class and II expression on respiratory epithelial cells involvement of a STAT1 and CIITA independent pathway J Virol 73 1411 Gao J B P Dev Han S Choudhary R Ransohoff and A K Banerjee 2001 Human parainfluenza virus type 3 inhibits yinterferon induced major histocompatibility complex class Il expression directly and by inducing a interferon J Virol 75 1124 Gnjatic S Y Nagata E Jager E Stockert S Shankara B L Roberts G P Mazzara S Y Lee P R Dunbar B Dupont V Cerundolo G Ritter Y T Chen A Knuth and L J Old 2000 Strategy for monitoring T cell responses to NY ESO 1 in patients with any HLA class allele PNAS 97 10917 Gordadze A V R Peng J Tan G Liu R Sutton B Kempkes G W Bornkamm and P D Ling 2001 Notch1IC
146. on at 100 This provides us with the following compensation matrix FITC PE FITC n a 5 00 PE 30 00 n a CyAn ADP User Guide 55 Assuming perfect expression the following particles represent all possibilities for FITC and PE expression within a population Particle FITC PE FlTCmeasure FiTCtrue PEmeasure PEtrue 1 100 100 100 100 2 1000 1000 370 100 3 145 100 1000 1000 4 1045 1000 1270 1000 Using the historical calculation of compensation where we subtract percentages from the measured values not the true values we incorrectly reach FITC true FITC measured 0 05 PE measured PE true PE measured 0 30 FITC measured Historical Historical Historical Historical Particle FITC RE FlTCmeasure FiTCtrue FlTCcomp Diffftrue comp PEmeasure PEtrue PEcomp Diffftrue comp 1 100 100 100 D 100 100 100 D 2 1000 1000 986 5 13 5 370 100 100 0 3 145 100 100 D 1000 1000 986 5 13 5 4 1045 1000 986 5 13 5 1270 1000 986 5 135 Here we see differences between the computed value and the true value which is entirely based on subtracting an percentage from a dirty value the raw measured value We term this dirty because it varies for each particle While particles 2 and 4 both have a true value of 1000 one measured at 1000 and the other at 1045 a 4 5 shift This suggests one reason why uncompensated histograms show population spreading that aren t biologically or physically tru
147. onses of progenitor B cells in human bone marrow through heterologous desensitization of the CXCR4 chemokine receptor Blood 100 2321 Jin L P A McLean B G Neel and H H Wortis 2002 Sialic acid binding domains of CD22 are required for negative regulation of B cell receptor signaling J Exp Med 195 1199 Kouro T K L Medina K Oritani and P W Kincade 2001 Characteristics of early murine B lymphocyte precursors and their direct sensitivity to negative regulators Blood 97 9 2708 2715 Lu S S J Tung N Baumgarth O Herman M Gleimer L A Herzenberg and L A Herzenberg 2002 Identification of a germ line pro B cell subset that distinguishes the fetal neonatal from the adult B cell development pathway PNAS 99 5 3007 3012 Marchbank K J C C Watson D F Ritsema and V M Hollers 2000 Expression of human complement receptor 2 CR2 CD21 in Cr2 mice restores humoral immune function J Immunol 165 2354 Martin F A M Oliver and J F Kearney 2001 Marginal zone and B1 B cells unite in the early response against T independent blood borne particulate antigens Immunity 14 5 617 629 Martin F and J F Kearney 2000 Positive selection from newly formed to marginal zone B cells depends on the rate of clonal production CD19 and btk Immunity 12 39 Miller J P D Izon W DeMuth R Gerstein A Bhandoola and D Allman 2002 The earliest step in B lineage differentiation from common lymphoid progenitors
148. or sample is run you will notice a negative population within the lower left quadrant with the FITC positive population occurring in the lower right quadrant The negative population occurs because not all cells within the lymphocyte population express the CD3 marker and thus will report as FITC Also you will notice that in the FITC versus PE plot the positive population is shifted into the upper right quadrant This means that the signal from the FITC population is also being detected by the PE channel The degree to which signal is being detected for the PE parameter represents the spillover of PE into the FITC signal G1 R1 FITC 0 amp 1 10000 5233 2207 7031 100 00 1 on oo 000 6 0 000 2697 3809 36 0 36 52 0 nao 2321 327 274 352 202 3279 2062 2912 523 2054 4759 67 21 count 2 7031 100 00 o ngo 0 Con 2322 3279 A13 Gm Ca 4759 6721 76 CyAn ADP User Guide dakocytomation Setting Compensation For proper compensation check to see that the median fluorescence for the populations within the lower two quadrants are equivalent Within 1 0 is typically acceptable but more accuracy is usually obtainable when performing compensation post acquisition If the values are not almost identical adjust the compensation settings such that the median fluorescent values of the FITC negative and positive populations are almost identical For this example compensation needed to be applied for th
149. overlap between the two signals the FITC and PE positive populations will appear offset that is to have a different median fluorescence from the negative population A FITC positive population which is not stained with PE will still appear slightly brighter in the PE parameter because of the spectral contamination from the FITC The same is true for FITC when running a PE single positive control This spectral overlap is what is physically observed by the cytometer by the photodetectors for each parameter The observed double negative The observed double negative and FITC positive populations and PE positive populations The degree that both populations are offset with regard to the ideal position represents the spectral overlap from one fluorescent signal into the other The observed fluorescent spillover The observed fluorescent spillover of FITC into the PE parameter of PE into the FITC parameter CyAn ADP User Guide 61 Keep in mind that the degree of spectral overlap for the two populations is not identical This is accounted for by the fact that the degree of overlap between the emission spectra of the two fluorescent compounds is not symmetrical Because of the spectral overlap between the FITC and PE signals compensation is needed to correct for the unwanted overlap or spectral cross contamination Compensation controls allow you to adjust or shift the data to mathematically remove the fluorescent overlap PE compen
150. pensation constants on markers that are physically not correlated The only advantage of this historical calculation is ease of implementation in analog circuitry on older flow cytometers The historical calculation for compensation can be described as a reasonable approximation albeit one that introduces error and creates anomalous mathematical sub populations that do not biologically or physically exist Pragmatic justifications for this technique fade with the advancement of new hardware and software and this approach can be completely replaced with the proper compensation algorithm 46 CyAn ADP User Guide JOB that has no such side effects Benefits include increased sensitivity on all experiments using fluorescent compensation and most especially with experiments utilizing an increasing number of colors Further pragmatic approaches to implementing proper compensation including iterative determination of compensation constants and compensation matrix inversion make proper compensation very reasonable to implement in both hardware and software Negative Compensation The Stanford Laboratories first broached the subject of negative compensation which remains quite controversial Just as many even experienced researchers and operators have a misunderstanding of compensation in general there remains a widespread misunderstanding of negative compensation and why it may be considered necessary by some scientists In short negat
151. r diode 635nm LE low CelCuvette Focusing Lens Figure 3 3 CyAn ADP UV Model Optical Block Diagram and Location of Laser Apertures 12 CyAn ADP User Guide Ge DakoCytomation The focused beams are separated vertically to ensure minimal optical crosstalk between detection paths Further the signals that are detected as particles that traverse the beams are electronically gated to reduce unwanted signal interference between channels A high numerical aperture microscope objective is used to collect scatter and fluorescence and to generate an image of the interrogation point for each of three apertures spatial filters The apertures improve signal to noise by preventing unwanted scatter around the excitation region from entering the detector block Light that transmits through the spatial filters is reflected and spectrally filtered on its way to the appropriate photomultiplier tubes PMT for detection CyAn ADP User Guide 13 Laser 1 H H KR j sf sbi oe d ch Gi P ee Filters 1 95 45DLP 95DLP 40DLP irror Dichroic Filters 730DLP mirror 425DLP f mirror Emission Filters lt lt _ 650DLP off le off aE BR T from E Point Filter configurations are easily modified to accommodate different applications Laser 3 Laser 2 Figure 3 4 CyAn ADP UV Model Detector Block and Optical Filter Layout Figures 3 4 and 3 5 illustrate the location position and orientation of the opt
152. r or LAN connection for downloading software updates Dimensions not including Auxiliary Height Components front cover closed 39 1 cm 15 4 in front cover open 72 1 cm 28 4 in Width 33 3 cm 13 1 in Depth 49 8 cm 19 6 in with clearance for cables 62 5 cm 24 6 in Weight 36 3 kg 80 Ibs Auxiliary Components CyAn ADP User Guide 5 Sheath Management System with casters Houses sheath container waste container cleaner fluid container air compressor and vacuum Height 61 3 cm 24 2 in Width 73 3 cm 28 9 in Depth 61 9 cm 24 4 in Weight 22 6 kg 50 Ibs Summit Workstation Height 42 9 cm 16 9 in Width 19 1 cm 7 5 in Depth 45 7 cm 18 0 in Weight 10 5 kg 23 Ibs Uninterruptible Power Supply Height 20 3 cm 7 9 in Width 14 7 cm 5 7 in Depth 44 5 cm 17 5 in Weight 20 kg 44 Ibs Operating Environment Ambient Temperature 15 to 30 C 59 to 86 F For optimum performance maintain at 2 C Relative Humidity 20 to 80 RH non condensing CyAn ADP Installation Requirements UV Model General Laboratory Information Heating and air conditioning vents or fans are not recommended directly above the CyAn ADP because of the resulting temperature fluctuation vibration and possible dust Table 2 2 CyAn ADP Installation Requirements UV Model General Requirements Po
153. require preparation of n 2 tubes The tubes for a 4 color experiment are defined above n unstained nonspecific isotype is best all stains sample Autofluorescence is a background level of fluorescence emitted from unstained and stained cells that is primarily due to intracellular flavins The effects of autofluorescence can be minimized by defining a region in the light scatter FSC vs SSC bivariate histogram and gating all of the fluorescence histograms from this population You should always gate on cells using light scatter before adjusting compensation to prevent autofluorescence After the tubes have been prepared proceed by running the unstained cells to set the PMT voltages as outlined below Using the negative isotype or unstained cells set the PMT voltages for each fluorescence channel so the population falls in the first decade log scale Always set Fluorescence ADC s to Log mode when setting up compensation and analyzing Using the negative tube plot a univariate Log scale histogram of the first stain channel and adjust the PMT voltage until the peak appears centered in the first log decade of the histogram Once this PMT voltage is set and compensation has been optimized it should not be changed throughout the experiment Now you can select a second parameter by simply right clicking on the X axis of the same histogram and selecting a different parameter Adjust the PMT of this parameter in the same way as t
154. ri B Scott P Hertzog L Tatarczuch and K Shortman 2002 Mouse plasmacytoid cells Long lived cells heterogeneous in surface phenotype and function that differentiate into CD8 dendritic cells only after microbial stimulus J Exp Med 196 1307 O Keeffe M H Hochrein D Vremec B Scott P Hertzog L Tatarczuch and K Shortman 2002 Dendritic cell precursor populations of mouse blood Identification of the murine homologues of human blood plasmacytoid pre DC2 and CD11c DC1 precursors Blood 2002 Qu C T M Moran and G J Randolph 2003 Autocrine type IFN and contact with endothelium promote the presentation of influenza A virus by monocyte derived APC J Immunol 170 1010 Reis e Sousa C G Yap O Schulz N Rogers M Schito J Aliberti S Hieny and A Sher 1999 Paralysis of dendritic cell IL 12 production by microbial products prevents infection induced immunopathology mmunity 11 637 Riese R J G P Shi J Villadangos D Stetson C Driessen A M Lennon Dumenil C L Chu Y Naumov S M Behar H Ploegh R Locksley and H A Chapman 2001 Regulation of CD1 function and NK1 1 T cell selection and maturation by cathepsin S Immunity 15 909 Schaniel C L Bruno F Melchers and A G Rolink 2002 Multiple hematopoietic cell lineages develop in vivo from transplanted Pax5 deficient pre B l cell clones Blood 99 472 136 CyAn ADP User Guide Gi oCytomation Vandenabeele S H Hochrein
155. rical compensation formula is based on pragmatics including real time processing constraints decisions during acquisition available hardware and technical hardware limitations and software availability For example fluorescent compensation has been achieved through analog subtraction in hardware where complex computation is not possible and only the computed value is reported not the raw value and in software where the input is the raw values reported by the instrument and software calculates the compensated value In extreme cases some users have performed both hardware and software compensation which can produce an apparently reasonable approximate compensated value although the validity of such a dual approach is always mathematically ambiguous Further simply correcting the constants as compensation coefficients or spillover coefficients is not sufficient The compensated value remains ambiguous unless the true value of the contaminating marker is used not the measured value This ambiguity is based on the fact that two particles with the same true value will have very different measured values cumulatively dependant on the presence of other markers on that particle The result of this mathematical ambiguity is population spread after compensation as illustrated in the example below Justifying a Different Compensation Algorithm Two Color Example It is mathematically ambiguous and technically incorrect to subtract
156. rine hematopoietic stem cell distribution and proliferation in ablated and nonablated bone marrow transplantation Blood 100 3521 T Cells Allenspach E J P Cullinan J Tong Q Tang A G Tesciuba J L Cannon S M Takahashi R Morgan J K Burkhardt and A Sperling 2001 ERM dependent movement of CD43 defines a novel protein complex distal to the immunological synapse Immunity 15 739 Avitahl N S Winandy C Friedrich B Jones Y Ge and K Georgopoulos 1999 Ikaros sets thresholds for T cell 142 CyAn ADP User Guide Gi oCytomation activation and regulates chromosome propagation mmunity 10 333 Bachmann M F A Gallimore S Linkert V Cerundolo A Lanzavecchia M Kopf and A Viola 1999 Developmental regulation of Lck targeting to the CD8 coreceptor controls signaling in naive and memory T cells J Exp Med 189 1527 Baumgarth N G C Jager O C Herman and L A Herzenberg 2000 CD4 T cells derived from B cell deficient mice inhibit the establishment of peripheral B cell pools PNAS 97 4766 Baur N G Nerz A Nil and K Eichmann 2001 Expression and selection of productively rearranged TCR B VDJ genes are sequentially regulated by CD3 signaling in the development of NK1 1 aT cells Int Immunol 13 1031 Briken V R M Jackman G F M Watts R A Rogers and S A Porcelli 2000 Human CD1b and CD1c Isoforms Survey Different Intracellular Compartments for the Presentation of Microbial L
157. s The CyAn ADP High Performance Flow Cytometer is a research tool engineered for precision analysis of cells bacteria and other similarly sized particles With CyAn ADP DakoCytomation sets a new industry standard with a combination of features never before available on a bench top analyzer CyAn ADP gives users three excitation lines with independent alignment free focusing optics simultaneous 9 color and 2 scatter parameters analysis rates of 50 000 events per second a full 9 x 9 interlaser compensation matrix and high sensitivity The result is stable user friendly and flexible technology The instrument is optimized for cell cycle kinetics fluorescent protein work and multi color immunophenotyping Rare event analysis such as MHC Dextramers Tdex studies and no lyse whole blood applications are easily performed on the CyAn ADP The instrument also provides simplified compensation before during and after acquisition with unequaled sensitivity in all fluorescent channels The main features of CyAn ADP include Walk Up Operation CyAn ADP offers walk up push button operation that puts even a novice user at ease With no optical alignment necessary the user simply places a 5 mL sample tube in the tube holder and chooses the desired protocol in Summit Software The software is used to control instrument settings Bench Top Configuration The CyAn ADP was designed with today s laboratories in mind The CyAn ADP measures 30
158. sation Matrix Helpful Te 46 Historical Compensation Calculation ccccececeecceececeeeeeeeceecaeceeeeesetecaaaeaeeeeeeesetensacaeeeeees 46 Negative COmpenSalions sosorry iser a A A duantagea soantecea hdcaen Suandege staves 47 Why Negative Compensation ccccececeeeeeeeeeeeeeeeteneeeeeeeeeeeeeaeeeeesaeeeeseneeeeeetnaeeeeseaes 47 A Negative Compensation Exvample nnanet ent 48 An Alternative to Negative Compensaton eter ee etieeeeetiieeeereiaeeeeetieeeeeeaa 51 Absolutely Perfect Compensation 0 cccceccececeeeeteeeeeetieeeeeenaeeeeeeceeeeeesneeeeetnaeeeeeeaas 52 2 Color Compensation EEN 54 Calculating Compensation Values 0 0 0 0 ceeeceeeeeeeeeeeeeeeeeeeeneeeeeeaeeeeeeaeeeeeenaeeeeeeenaeeeeeenaes 54 Justifying a Different Compensation Algorithm Two Color Exvample A 55 2 Color Compensation Experiment 57 Goal of Compensation edd aie eee iaaa ae deadlines 58 How does this work with Dot Plots 0 cccecceeeecne cece eeeeeeenecaeceeeeeeesececaeeeeeeeeetenseaeees 59 An Example Using Fluorescent Beads 0 cccccceeeeeeeeeeeeeeteeeeetineeeeeseeeeeetiieeeeeesneeeeeeaa 64 Compensating the Data seisein iioii aeania Niria nE erii a a 64 Advanced SSUGS anian ea aa a sae eee aes oe eee eee 67 3 Color Compensation DEE 68 Calculating 3 Color Compensation Values 69 Proper Compensation Pragmatics cccccccccecccecceceeeeeeeeeeeaeeeeeeeeeseccanaeeeeeeseeeeesnnnaeees 70 5 Color Compensation Experi
159. sation eliminates FITC FITC compensation eliminates PE contribution to the PE signal contribution to the FITC signal nd 62 CyAn ADP User Guide Ge DakoCytomation Compensation is adjusted to align populations so that the median fluorescence in the stained population is equivalent to the median fluorescence in the unstained population Positioning of the data is performed with respect to the negative control population A PE negative population should have the same median value as all the other PE negative populations regardless of the presence of FITC receptors on that population Equivalent median PE fluorescence between Fquivalent median FITC fluorescence betwen the FITC positive and negative populations the PE positive and negative populations eo ee Typically after the data is compensated the populations will appear more stretched or oblong because the population is moving along a logarithmic axis For example on a logarithmic scale the difference between two channels at the beginning of the first decade is one the difference between two channels at the beginning of the second decade is ten and the difference between two channels at the beginning of the third decade is one hundred When the population is pulled lower on the axis the population retains the same resolution but the data appears to stretch Further compensation algorithms used by some software may stretch the population because of the compensation
160. sion in a new ROSAZ26 reporter mouse strain Blood 97 1 324 CyAn ADP User Guide 137 McGavin M K H K Badour L A Hardy T J Kubiseksi J Zhang and K A Siminovitch 2001 The intersectin 2 adaptor links Wiskott Aldrich Syndrome protein WASp mediated actin polymerization to T cell antigen receptor endocytosis J Exp Med 194 12 1777 1787 Mohrs M K Shinkai K Mohrs and R M Locksley 2001 Analysis of type 2 immunity in vivo with bicistronic IL 4 reporter mmunity 15 2 303 311 Monroe R J K J Seidl F Gaertner S Han F Chen J Sekiguchi J Wang R Ferrini L Davidson G Kelsoe and F W Alt 1999 RAG2 GFP knockin mice reveal novel aspects of RAG2 expression in primary and peripheral lymphoid tissues Immunity 11 201 212 Nakamura K A Malykhin and K M Coggeshall 2002 The Src homology 2 domain containing inositol 5 phosphatase negatively regulates Fcy receptor mediated phagocytosis through immunoreceptor tyrosine based activation motif bearing phagocytic receptors Blood 100 9 3374 3382 Olaso E K Ikeda F J Eng L Xu L Wang H C Lin and S L Friedman 2001 DDR2 receptor promotes MMP 2 mediated proliferation and invasion by hepatic stellate cells J Clin Investigation 108 9 1369 1378 Rada C J M Jarvis and C Milstein 2002 AID GFP chimeric protein increases hypermutation of Ig genes with no evidence of nuclear localization PNAS 99 10 7003 7008 Rommel C B A Clarke S Zim
161. sitive negative response for each of the three colors Assume that 10 is the value for a positive response we saw those photons for that color and 0 is the value for a negative response we did not see any photons for that color Real Values if no spectral overlap Event Yellow Green Blue 1 D 0 D 2 10 0 D 3 0 10 D 4 0 0 10 5 10 0 10 6 10 10 D 7 0 10 10 8 10 10 10 The table above notes biological reality there is truly no Green response on Event 2 because that event was negative for Green However because we stated that there is a 30 spectral overlap for neighboring colors we will actually observe the following table where a little of each positive color washes into the next color For example Note that Event 2 now shows a small amount of Green because it was positive for Yellow and there was 30 carry over to the Green spectrum the other events behave similarly 48 CyAn ADP User Guide Observed Values Event Yellow Green Blue 1 0 0 0 2 10 3 H 5 3 10 3 4 0 3 10 5 10 6 10 6 13 13 3 d 3 13 13 8 13 16 13 Note Typically the operator will run controls for each color and adjust the PMTs to obtain some positive value for a given color thus it will be tuned to provide a positive 10 for each color Thus any carry over to the next color 30 in this example does not detract from the positive value that was set when the PMTs were calibrated for the experiment We now have the typical problem where
162. tes does not require T cell receptor major histocompatibility complex contact J Exp Med 190 757 Felix N J W J Brickey R Griffiths J Zhang L Van Kaer T Coffman and J P Ting 2000 H2 DMa mice show the importance of major histocompatibility complex bound peptide in cardiac allograft rejection J Exp Med 192 31 Fiorini E Schmitz W E Marissen S L Osborn M Touma T Sasada P A Reche E V Tibaldi R E Hussey A M Kruisbeek E L Reinherz and L K Clayton 2002 Peptide induced negative selection of thymocytes activates transcription of an NF B inhibitor Mol Cell 9 637 Fowell D J K Shinkai X C Liao A M Beebe R L Coffman D R Littman and R M Locksley 1999 Impaired NFATc translocation and failure of Th2 development in Itk deficient CD4 T cells Immunity 11 399 Gartner F F W Alt R Monroe M Chu B P Sleckman L Davidson and W Swat 1999 Immature thymocytes employ distinct signaling pathways for allelic exclusion versus differentiation and expansion Immunity 10 537 Ghia P P Transidico J P Veiga C Schaniel F Sallusto K Matsushima S E Sallan A G Rolink A Mantovani L M Nadler and A A Cardoso 2001 Chemoattractants MDC and TARC are secreted by malignant B cell precursors following CD40 ligation and support the migration of leukemia specific T cells Blood 98 533 Gibbons D N C Douglas D F Barber Q Liu R Sullo L Geng H J Fehling
163. that in this case the calculated positives differ from the first example control Sample Positives CyAn ADP User Guide 117 In practice real data sets involve many more events per channel but the same mathematical principles still apply The following illustrates a subtraction histogram containing a single sample with a control In this example the positive population is identified as the following Ei Subtraction FL 1 1284 963 Control Positives If the sample and control in this experiment are reversed the following positive population is calculated EI Subtraction FL 1 Sample Control Positives 118 CyAn ADP User Guide Gi DakoCytomation The Overton method calculates positive events on a percentage basis as a function of reverse cumulative distribution This means that positive events reported within each sample channel are calculated by subtracting a number of control events based on that channel and all higher channels Ultimately this method helps to reduce the number of false positives and better reflects true biological differences For example using the Overton method below the following positive population would be identified In this case the result is the same as with the channel method Subtraction FL 1 1264 963 Positives However if the sample and control assignments are reversed no positives are reported with the Overton method Using the channel method positives are
164. the data s event count in various channels would eventually exceed the set Y axis count At that point peak values in the data are not visible and the data would be defined as offscale Ei Data Scaling Example AN By Data Scaling Example 79 With auto scaling when the number of events in the peak data channel approach the histogram s count limit the Y axis is automatically rescaled to encompass a greater count range In turn the displayed data is resized based on the new Y axis count As acquisition continues the process of rescaling the Y axis and resizing the data is repeated each time the data reaches the histogram s count limit This ensures that the full data display remains in the histogram and does not drift offscale CyAn ADP User Guide 111 Ei Data Scaling Example lt 7 UL Data Scaling Example 79 Auto Scaling Options There are a number of settings that can be adjusted for auto scaling Auto Scale Teale Hit Value ER ale Increment by 50 E Ignore Lower z Channels Ignore Upper z H Channels Hit Value refers to the maximum height or of counts that the data s peak can reach in the histogram before the Y axis is rescaled B Data Scaling Example 160 Hit Value 90 Increment by determines the degree by which the Y axis is adjusted and the data is resized The scale is incremented when the data s peak count reaches the assigned hit value in the histogram 112 CyA
165. tinse Sheath Management System LG A Laser LG amp Interlock e P Press Es Vac Figure 5 1 SMS section of Control Panel If an error condition occurs or status changes for a subsystem an indicator light will change from green to amber or red When you place your cursor over the indicator light a message appears that provides a description of the warning and instructions for how to fix the problem The following figures illustrate some of the possible error conditions in the SMS CyAn ADP User Guide 27 Sheath Management System Message 410 Less than 10 minutes of sheath fluid is remaining Please replenish your sheath fluid Figure 5 2 Low sheath fluid warning Sheath Management System M d Figure 5 4 Cleaner quick connect error message 28 CyAn ADP User Guide dakocytomation The LED indicators on the front panel of the Sheath Management System also change when fluid levels change in the SMS Three fluid levels are monitored on the SMS panel SHEATH LEVEL CLEANER LEVEL and WASTE LEVEL When all fluids are at suitable levels for proper operation of the CyAn ADP the SHEATH LEVEL CLEANER LEVEL and WASTE LEVEL LEDs appear green beside the OK label SHEATH MANAGEMENT SYSTEM RESET CLEANER LEVEL WASTE LEVEL TO DakoCytomation When the sheath level or cleaner fluid level is low the green OK LED changes to a flashing amber or a solid amber LED beside the LOW label
166. tion Field Service Representative should perform an annual maintenance check on the CyAn ADP To schedule an annual maintenance check contact your local DakoCytomation Support Representative 36 CyAn ADP User Guide M dak SECTION 7 TROUBLESHOOTING There are no operator replaceable fuses in the CyAn ADP Contact your local DakoCytomation Field Service Representative immediately for assistance with any instrument malfunction or service need WARNING Do not attempt any maintenance on the CyAn ADP laser components Laser maintenance should only be performed by specially trained certified DakoCytomation Field Service Representatives The following table is a guide for troubleshooting CyAn ADP problems Table 6 1 CyAn ADP Troubleshooting Guide Problem Action Vacuum pressure on the waste container is Make sure the quick connects are connected low If still low contact customer service if Vacuum No sheath pressure Make sure that the quick connects are fully engaged it Pressure If there is still no pressure contact customer service CyAn has no power Make sure that the power plug is firmly attached to the wall Make sure that the power switch on the back of the CyAn is on Acquiring data but no display Make sure that the lasers are on Make sure that the laser shutters are open on the CyAn Control Panel CyAn ADP User Guide 37 38 CyAn ADP User Guide SECTION 8 TUTORIALS Using C
167. tive Maintenance 35 Laser Interlock Maintenance Procedure sssseesrranssrnsseernannnnnnneennaannannantennaanannaneennaanana 35 Annual Maintenance Procedure 2edgeiCedkgeteERdER REESEN ENEE EAEE ANAKNA ARANARAK 36 SOQCUOMN E 37 TIOUDIESNOOUM GE esien E OEA nei tlnbe nee T 37 Section E 39 CyAn ADP User Guide vii viii T t nalS EE eae aerate panne trate Maes ce cheat re he eal a a 39 Eine Rule Le EE 39 The argument against COMPENSATION eee eee eeeeeeeeete eect eeteeeeettaeeeeeeeaeeeeetaeeeeeetaeeeeeeaes 39 The argument for Compensaton eee eeeeeeeeeetee ee erent ee eetaeeeeetaeeeeeeaaeeeeeenaeeeeeenaeeeeneaes 39 Hardware vs Software Compensation ssssseseeenserssteetttrtttssttttttnntttstttnr tn nnn nnar tt nnne nenene 40 Hardware Compensation eene aie e tiaa ai aa aie A 40 Software Compensation tnia isores daii eee aa a enebi eei a a ee RA 41 Digital Signal Processing DSP Compensaton 42 Fluorescent Compensation c ccccecnececeeeeeeeceeeeeeeeeeesecaaaaeaeeeeeeesesaneaeaeeeeeeesecsecaesaeeeeeess 43 Background Use in Flow Cytometry essssessserseesrieeesinnesnnrnnaitnneettnnaaeinndennnnaannnneetannnaeana 43 Fluorescent Cross Contamination in the Raw Measure 43 Compensation Preparation cccccceceeeeeceeceeeeeseeeeaeeeeeeeeesecceaaaeaeeeeeeeseseccueeeeeeeeeenesenaeees 45 Preparation SUMMARY 35 2 ess a cant cneet create ecaauet inne EA EAE REE A 45 Completing the Compen
168. ublets using Color Gang 106 Methods of Doublet Discrimination ccccceeeeceeeeeeeeeeeneeeeeeneeeeeeeaeeeeeeaeeeeeetaneeeeeaes 107 Histogram Scaling ceeecceeeeeeeee eect ee erent eee seen ee erent ee erent ee eee aeeeeeeaaeeeeeeaaeeeeeeaeeeeesneeeesas 108 Manual Scaling ceeeeeeeeeee tenner ee eeeneeeeeeeaeeeceeeeaeeeseeeaeeeeeeaaeeeeeeaaeeeeeeaeeeeeeeeeseeeneeeeseaas 109 Manual Scaling Optons tatt ttu tantun unatu unnt nn unat nunnat nn nannten nat 109 increment Bye srcriorarceciiiairiniiarii E E aE EAEEREN A AREA 109 Set Fixed Scale 2a ee ee ee eee eee 110 PAULO SCAN Gerea i ceeds vice un beeen dpbageiae deagedack T T A A 111 Auto Scaling ee E 112 leiere Be tee 114 Controls Versus Samples sssossesnieneseeeettrttnsttertttntnnsttertntnntasstertttn nnne st ten ttnnnnnenen ennn nn ne 114 Subtraction une 116 Experimental and Biological Relevance sesssesssrrsseernasssnnaseennaannnnnneennaanannaaatennaanannaaeena 120 Nagoen Tt E 123 GYAN ADP Consumables er aaa E E EEE E OEE 123 ee Tt a EE 125 Technical and Instrument Specifications ccccccceeeeeeeeceeceeeeeeeeeeeeeeeeaeeeeeeeseseceieaeeeeeeeeeteees 125 PADD OMNI KC recess be E E suuacuauyacenns suuaaaaayaceens caeusunnoeseeendateneaaneae 133 Flow Cytometry Referenc s 1 decl easel ted ETE EEEE 133 CyAn ADP User Guide ix CyAn ADP User Guide gt be P CyAn ADP User Guide Mos oCytomation SECTION 1 SAFETY Electrical Safety
169. ummit Workstation 39 1cm 15 4 in front cover closed 72 1cm 28 4 in front cover open Width 119 4cm 47 0 in 132 1cm 52 0 in including clearance for cables Depth 59 2cm 23 3 in Weight 86 5 kg 190 Ibs Dimensions not including Utility Cart or Summit Workstation Height 39 1cm 15 4 in front cover closed 72 1cm 28 4 in front cover open Width 33 3cm 13 1 in Depth 49 8cm 19 6 in 62 5cm 24 6 in cables including clearance for Weight 36 3 kg 80 Ibs Auxiliary Components Laser Power Supply UV Model Height 19 cm 7 5 in Width 43 cm 17 in Depth 61 cm 24 in Weight 31 kg 68 Ibs Sheath Management System with casters Houses sheath container waste container cleaner fluid container air compressor laser power supply and vacuum Height 61 3 cm 24 2 in Width 73 3 cm 28 9 in Depth 61 9 cm 24 4 in Weight 52 kg 115 Ibs Uninterruptible Power Supply Height 20 3 cm 7 9 in Width 14 7 cm 5 7 in Depth 44 5 cm 17 5 in Weight 20 kg 44 Ibs CyAn ADP User Guide 129 Summit Workstation Height 42 9cm 16 9 in Width 19 1cm 7 5 in Depth 45 7cm 18 0 in Weight 10 5 kg 23 Ibs External Transformer Height 19 1cm 7 5 in Width 31 2 cm 12 3 in Depth 19 1 cm 7 5 in Weight 11 4 kg 25 Ibs Safety Interlock The front cover is protected with dual
170. us PE Cy5 plots these cells will run in the upper left quadrant indicating a positive signal for PE Cy5 In the PE versus FITC plot these cells run in the lower left quadrant indicated that they are negative for both FITC and PE 2 G1 RI All Stains M E3 G1 R1 All Stains BRT ES fi G1 91 All Stans Oo x u E GI a PE Cy5 Comp PE Cy5 Comp FITC Comp viet LTH e 1 101 10 107 10t A D 10 10 107 10t FITS Corre PE Corres Non T or B cells Le NK cells Non T or E cells i e NK cells Non T or E cells i e NK cells CD45 PE Cy5 CD45 PE CyS CD45 PE Cy5 cD3 FITC CD3 FITC cD3 FITC CD19 FE CD19 PE cD19 PE To better identify those cells that are not T or B cells a complex gating strategy is used A gate can be created to include the quadrants where these cells should fall the upper left quadrant for the FITC versus PE Cy5 and PE versus PE Cy5 plots and the lower left quadrant for the PE versus FITC plot By applying this gate to all three plots only those cells that occur in all 3 90 CyAn ADP User Guide dakocytomation regions will be displayed Additionally the gate for region R1 is also included Below illustrates how by applying a gate that includes regions R1 R24 R8 and R2 the non T and B cells i e NK cells can be separated from other lymphocyte cells L GB R24 amp R1 amp R8 amp R2 All Stains
171. utation won t be necessary except to speed your data display during acquisition 42 CyAn ADP User Guide CB oCytomation Fluorescent Compensation Background Use in Flow Cytometry Flow cytometry performs measurement of multiple parameters on an individual particle usually as that particle passes one or more lasers at one or more interrogation points An individual sample may contain many particles and these particles are analyzed individually with many parameters for each particle Thus a tremendous amount of flow cytometric data is typically created for each sample for each collection of particles Some measurements such as particle size and shape can be determined from pulse width and light scatter patterns as the particle passes a laser However it is common to treat particles with reagent markers that bind with some level of selectivity to proteins large molecules or other cell structures when these markers fluoresce after excitation by a laser with a given power and frequency range the resulting measure is used to indicate particle attributes Fluorescent Cross Contamination in the Raw Measure Commonly more than one fluorescent marker is used in a single experiment and an individual particle will express more or less of one or the other or both markers Below is an example of the spectral emission wavelength for two such markers commonly used together FITC and PhycoErithryn PE It is clear to see that some spectral overlap
172. volvement from the institutional or company laser safety officer DakoCytomation does not provide regulatory certification for the individual lab All access plates and other user accessible points of potential laser exposure are clearly designated on the CyAn ADP instrument with the labels illustrated in figures 1 1 1 2 and 1 3 below Interlocks are designed to prevent accidental irradiation of the operator The user should not defeat these interlocks Figure 1 1 Laser Warning Label 2 CyAn ADP User Guide DakoCytomation Figure 1 3 Laser Warning on Rear of CyAn Unit CyAn ADP User Guide 3 CyAn ADP User Guide 0 SECTION 2 INSTALLATION CyAn ADP Installation Requirements IMPORTANT Your DakoCytomation representative is responsible for uncrating installing and initial set up of the CyAn ADP General Laboratory Information Heating and air conditioning vents or fans are not recommended directly above the CyAn ADP because of the resulting temperature fluctuation vibration and possible dust Table 2 1 CyAn ADP Installation Requirements General Requirements Power Requirements 100V 240V 600VA W 50 60 Hz Lab Bench Stable bench table top to support the CyAn ADP one or two monitors keyboard and a mouse pad Service Access 45 7 cm 18 in minimum around instrument components Phone Location near CyAn ADP for contacting technical support Internet Access Internet Service Provide
173. wer Requirements 100V 240V 600VA W 50 60 Hz Lab Bench Stable bench table top to support the CyAn ADP one or two monitors keyboard and a mouse pad Service Access 45 7 cm 18 in minimum around instrument components Phone Location near CyAn ADP for contacting technical support CyAn ADP User Guide G General Requirements Internet Access Internet Service Provider or LAN connection for downloading software updates Dimensions not including Auxiliary Components Height front cover closed 39 1 cm 15 4 in front cover open 72 1 cm 47 0 in Width 119 4 cm 13 1 in Depth 59 2 cm 23 3 in with clearance for cables 132 1 cm 52 0 in Weight 86 5 kg 190 Ibs Auxiliary Components Sheath Management System with casters Houses sheath container waste container cleaner fluid container air compressor and vacuum Height 61 3 cm 24 2 in Width 73 3 cm 28 9 in Depth 61 9 cm 24 4 in Weight 52 kg 115 Ibs Summit Workstation Height 42 9 cm 16 9 in Width 19 1 cm 7 5 in Depth 45 7 cm 18 0 in Weight 10 5 kg 23 Ibs Uninterruptible Power Supply Height 20 3 cm 7 9 in Width 14 7 cm 5 7 in Depth 44 5 cm 17 5 in Weight 20 kg 44 Ibs Operating Environment Ambient Temperature 15 to 30 C 59 to 86 F For optimum performance maintain at 2 C
174. within the first or lower channel and the other half within the second or upper channel For a symmetrically distributed CyAn ADP User Guide 99 population four channels would bin events equally among all 4 channels By increasing the channel number the granularity by which events can be distinguished from one another increases A data set distributed within 1 2 and 4 channels gt Symmetrical Distribution In reality when samples are run through a cytometer individually recorded signals are not likely to represent a perfect symmetrical distribution Individual events are assigned a channel based on the detected signal for that parameter and as such may not be displayed equally in all channels The example below illustrates how events from a single channel may be binned according to the actual detected signal intensities In the 2 channel situation rather than dividing up the signals equally i e half in one channel and half in the other a greater number of events may be assigned to one channel This distribution is based on the actual recorded signals In this case more appear in the left most channel since more of the events were detected with a lower signal intensity This unequal distribution is carried over and seen when the same events are distributed into 4 channels A data set distributed within 1 2 and 4 channels Real Distribution The same logic of binning data and channels applies to histograms
175. y increased proliferation Blood 99 3792 Lu X G Magrane C Yin D N Louis J Gray and T Van Dyke 2001 Selective inactivation of p53 facilitates mouse epithelial tumor progression without chromosomal instability Molecular and Cellular Biology 21 17 6017 6030 Rego E M Warrell R P Z G Wang and P P Pandolfi 2000 Retinoic acid and AsO treatment in transgenic models of acute promyelocytic leukemia unravel the distinct nature of the leukemogenic process induced by the PML RARa and PLZF RARa oncoproteins PNAS 97 18 10173 Schwieger M J Lohler J Friel M Scheller Horak and C Stocking 2002 AML1 ETO inhibits maturation of multiple lymphohematopoietic lineages and induces myeloblast transformation in synergy with ICSBP deficiency J Exp Med 196 1227 CyAn ADP User Guide 135 So C W and M L Cleary 2003 Common mechanism for oncogenic activation of MLL by forkhead family proteins Blood 101 633 Stripecke R A A Cardoso K A Pepper D C Skelton X J Yu L Mascarenhas K I Weinberg L M Nadler and D B Kohn 2000 Lentiviral vectors for efficient delivery of CD80 and granulocyte macrophage colony stimulating factor in human acute lymphoblastic leukemia and acute myeloid leukemia cells to induce antileukemic immune responses Blood 96 4 1317 1326 Tomasson MH I R Willliams S Li J Kutok D Cain S Gillessen G Dranoff R A Van Etten and D G Gilliland 2001 Induction of myeloproli
176. ytometry 36 340 348 Dorris D R and K Struhl 2000 Artificial recruitment of TFIID but not RNA polymerase II holoenzyme activates transcription in mammalian cells Molecular and Cellular Biology 20 12 4350 4358 Eischen C J M F Roussel S J Korsmeyer and J L Cleveland 2001 Bax loss impairs Myc induced apoptosis and circumvents the selection of p53 mutations during Myc mediated lymphomagenesis Molecular and Cellular Biology 21 22 7653 7662 Gartner F F W Alt R J Monroe and K J Seidl 2000 Antigen independent appearance of recombination activating gene RAG positive bone marrow B cells in the spleens of immunized mice J Exp Med 192 12 1745 1754 Kishimoto J R E Burgeson et al 2000 Wnt signaling maintains the hair inducing activity of the dermal papilla Genes amp Development 14 10 1181 1185 Kishimoto J R Ehama L Wu S Jiang N Jiang and R E Burgeson 1999 Selective activation of the versican promoter by epithelial mesenchymal interactions during hair follicle development PNAS 96 7336 7341 Lorens J B M K Bennett D M Pearsall W R Throndset A B Rossi R J Armstrong B P Fox E H Chan Y Luo E Masuda D A Ferrick D C Anderson D G Payan and G P Nolan 2000 Retroviral delivery of peptide modulators of cellular functions Molecular Therapy 1 5 438 447 Mao X Y Fujiwara A Chapdelaine H Yang and S H Orkin 2001 Activation of EGFP expression by Cre mediated exci

CyAn™ ADP User Guide - Weatherall Institute of Molecular Medicine

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CyAn&trade; ADP User Guide - Weatherall Institute of Molecular Medicine

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CyAn™ ADP User Guide - Weatherall Institute of Molecular Medicine