Muse™ PI3K Activation Dual Detection Kit User's Guide
Contents
1. B Statistics Isi tie nie Statistics Sample Info Mean Intensity Mean Total Intensity of Cells PISK Total of Cells Inactivated UL 69 40 8 16 Inactivated UL 69 40 8 16 J Select to hide plots Activated UR 11 80 Non expressing LL 12 50 Nori xpressing Neg PISK EXPRESSION Pos nactivated Activated inact PHOSPHORYLATION Act ulii Finish Next Run Select to display Beye Next Run plots Save amp close data set Run next sample You can view or change the sample ID as well as add annotations for the current sample by selecting the Sample Info Tab To print the results for the current sample select the printer tab 12 Optional If changes are needed to the gates assigned touch the dot plot to enlarge it then adjust the cell size gate according as described in steps 4 and 6 respectively You cannot adjust the cell size threshold after the sample has been acquired If you adjust the gate on subsequent samples and wish to apply the changes to other samples that you already acquired select the Apply Changes button in the title Select to apply bar Select the samples you want to apply the changes Traces Se COE to or choose Select All then select Apply The sample you originally made changes to must be selected z O W 0 x fr Y a 0 1 2 x Inact PHOSPHORYLATIO The qu2. Muse M PISK Activation Dual Detection Kit User s Guide Catalog No MCH200103 50 tests FOR RESEARCH USE ONLY Not for use in diagnostic procedures USA amp Canada Phone 1 800 437 7500 Fax 1 951 676 9209 Australia 61 3 9839 2000 www millipore com Introduction EMD Millipore s Muse Dual Detection kits are a series of products which include a pair of antibodies that bind to the same protein one to detect total protein expression and another to detect the phosphorylated form of the same target So by using two parameter analysis we can achieve target specific detection of phosphorylation and by doing so eliminate false positives while enhancing the signal to noise ratio Data generated using the Muse Cell Analyzer with the Muse software provides e Percentage of inactivated cells e Percentage of activated cells via phosphorylation e Percentage of non expressing cells Akt PKB is a Ser Thr kinase and a major known effecter of the PI3 Kinase pathway It is involved in multiple signaling pathways that relate to many biological processes including metabolism apoptosis cell cycle control angiogenesis differentiation and cell growth and proliferation In mammals three isoforms of Akt Akt1 PKCa Akt2 PKBB and Akt3 PKBy exists They exhibit a high degree of homology but differ slightly in the localization of their regulatory phosphorylation sites Akt1 is the predominant isoform that is in most tissues and is though
3. E rese place ce a as place eq a E 2 Move gi amp 2 Move gi E populati tg 5 populati ta kep FN Non exj i ae Non exg Oo o PHOSPHORYLATION 1 To set controls Back Back Back Next Set Threshold erify Settings Set Threshold Verify Setting Set Threshold Verify Settings Example 2 Human Jurkat cells Constitutively activated Adjust Settings Step 2 of 2 Inactivated j Inactivated PI3K EXPRESSION PI3K EXPRESSION PI3K EXPRESSION 0 1 2 3 PHOSPHORYLATION Act ha PHOSPHORYLATION Act h l Inact PHOSPHORYLATION Act POPULATION PROFILE 1 To set controls to POPULATION PROFILE 1 To set c 3 z a Z place C re ME machos 9 7 7 7 3 M 2 Move g a X ee wa x E populati te ndr EM ta N Non ex PHOSPHORYLATION PHOSPHORYLATI POPULATION PROFILE 1 To sete 7 Select Next when the marker adjustments are complete 8 Verify the settings If the settings are correct select Next Otherwise select Back and repeat steps 4 through 7 as necessary A For Mouse NIH3T3 cells B For Human Jurkat cells Verify Settings POPULATION PROFILE Inactivated Verify Settings POPULATION PROFILE PISK EXPRESSION Pos PISK EXPRESSION Pos PISK EXPRESSION Pos PISK EXPRESSION Pos Neg Neg Neg Neg 0 1 2 3 CELL SIZE INDEX Inset PHOSPHORYLATION Act CELL SIZE INDEX Inset PHOSPHORYLATION Act If settings are correct se
4. 5 minutes on ice 8 Spin down cells at 300 x g for 5 minutes in a tabletop centrifuge and discard supernatant 9 Permeabilize cells by adding 1 mL ice cold Permeabilization Buffer per one million cells and incubate on ice for 5 minutes For smaller cell samples t e 500 000 cells add 500 uL ice cold Permeabilization Buffer 10 Spin down cells at 300 x g for 5 minutes in a tabletop centrifuge and discard supernatant 11 Resuspend cells in 450 uL 1X Assay Buffer per one million cells and aliquot 90 uL per microcentrifuge tube Compatible for the Muse Cell Analyzer Please see Materials Not Supplied section on page 3 Ill Cell Staining and Analysis 12 For single color staining e g adjustment settings add 5 uL of anti Akt PKB PECy5 antibody to the microcentrifuge tube containing the cell suspension 13 For multiplexing add 10 pL of the antibody working cocktail solution as previously described to each microcentrifuge tube containing the cell suspension 14 Incubate cell testing samples for 30 minutes in the dark at room temperature 15 Following incubation step add 100 uL of 1x Assay Buffer to each microcentrifuge testing sample and centrifuge at 300 x g for 5 minutes on a tabletop centrifuge Discard supernatant 16 Resuspend cells in each microcentrifuge tube with 200 pL of 1x Assay Buffer 17 Acquire samples on the Muse Cell Analyzer using the onscreen instructions Setup and Acquisition on the Muse Cell A
5. cells prepare an antibody working cocktail solution by addition of the following Add 5 uL of anti phospho Akt Ser473 Alexa Fluor amp 555 and 5 uL of anti Akt PKB PECy5 conjugated antibodies into a centrifuge tube for a final volume of 10 uL total This amount is good for one 1 test Based on the number of tests tubes being performed it is up to the end user to adjust antibody volume amounts at similar ratios e g for 10 tests the working cocktail solution will contain 50 uL of anti phospho Akt Ser473 and 50 uL of anti Akt PKB for a total of 100 uL Aliquot 10 uL of the working cocktail solution per test tube sample This solution should be prepared as needed but if temporary storage is needed please keep in the dark at 2 8C Assay Instructions Note This assay protocol has been optimized using both mouse embryonic fibroblast NIH3 T3 cells and human jurkat cells However this kit is suitable for measuring the extent of PISK target specific detection of activation or deactivation via phosphorylation on a variety of human and mouse cell types in which Akt is expressed Alternate species reactivity must be confirmed by the end user I Cell Culture and Stimulation Used for example purposes 1 Prepare cells of interest into two tissue culture flasks treated or untreated overnight in a 37 C incubator with 596 CO2 Cells should be at about 9096 confluent the next day 2 For the flask labeled Treated treat cells accordingly
6. e g chemically treated using PDGF Wortmannin or compound of choice The intent is to induce Akt activation deactivation for the given cell type The other flask labeled untreated will serve as a control 3 Incubate the flasks in a 37 incubator with 5 CO2 Exposure time and treatment concentrations are determined at the discretion of the end user 4 Deactivate cells by exchanging out the growth media with fresh growth media or 1X PBS All cell treatments and experimental samples are determined by the end user This section is provided only as an example for inducing an Akt response for measurement of phospho Akt activation and or deactivation ll Fix and Permeabilize Cells 5 After cellular deactivation spin down the treated and untreated testing samples at 300 x g for 5 minutes and discard the media 6 Resuspend cells by adding 500 uL of 1X Assay Buffer per one million cells for larger cell samples i e 5x10 cells add 2 5 mL 1X Assay Buffer to cell sample Essentially add 50 uL of 1X Assay Buffer for every 100 000 cells evaluated 7 Add equal parts Fixation Buffer to cell suspension 1 1 So for every 500 uL of 1X Assay Buffer per one million cells add an additional 500 uL Fixation Buffer for a total of 1 mL cell fixation solution and mix sample by gently pipetting up and down Similarly add 50 pL of Fixation Buffer for every extra 100 000 cells evaluated to keep the 1 1 ratio consistent Incubate for
7. this phosphorylation event samples must be immediately activation in testing fixed to freeze the given activation state in time samples Low Cell Concentration Ensure that cells are counted properly prior to beginning the experiment warning during The assay instructions are optimized to give you a range of cells between 300 700 cells uL in the final sample volume so accurate population count results are obtained A substantial decrease in cell numbers can lead to difficulty in adjusting settings High Cell If the concentration of the stained cell sample is high 21200 cells uL dilute Concentration the sample further with Muse Cell Cycle Reagent to adjust the cell warning during concentration between 300 and 700 cells uL acquisition High CVs wide Although the assay procedure has been optimized to function for multiple peaks or false peak cells types every cell line behaves differently The wide peaks or false peak may indicate that e he sample is poorly fixed and stained as a result of cell aggregates Ensure your sample is a single cell suspension before fixing and staining Cell concentration is too high Decrease the number of cells by diluting the sample to 300 700 cells uL The Muse Cell Analyzer gives the most accurate data when the flow rate is between 300 and 700 cells uL Low level of staining Although the assay procedure has been optimized to function utilizing multiple cell types every cell line behaves differ
8. within the total cell population Cells which express Akt can be seen by the data on the top two quadrants of the dot plot inactivated and activated representing about 81 296 of the total cell population But of this cell population 69 4 is deactivated upon treatment attenuating the constitutive activation of the Akt signaling pathway in jurkat cells By presentation of both datasets one can now determine the total phospho ratio within their testing samples 14 Statistics Sample Info TT Statistics ESEI CE ae Mean PI3K Total of Cell o 023 Intensity ied PI3K Total Mean of Cells Intensity 0 Inactivated UL 21 10 9 90 211 Inactivated UL 21 10 9 90 211 Select plot to adjust marker gate POPULATION PROFILE 2 EXPRESSION Activated UR 73 80 32 08 738 Non expressing LL 1 50 3 99 15 50 Non expressing o 7 D Tm ta a gt lt i Y e a e 0 4 inact PHOSPHORYLATION Act Figures C and D NIH3T3 cells were exposed to 50 ng mL PDGF for 5 minutes at 37 to activate the Akt signaling cascade response fixed permeabilized and then stained with both anti phospho Akt Ser473 and anti Akt PKB antibodies in multiplex In a similar fashion samples were acquired using the Muse Cell Analyzer and statistical results are shown above Figure C shows the results summary while Figure D shows results displayed
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10. a calculating the expression of each cell population as a percentage Results from each run are stored in a data file as well as its corresponding spreadsheet CSV file The data file and spreadsheet file contain the following statistics Sample Number Sample ID Percent totals for the Inactivated Activated Non expressing cell types Mean Fluorescence Intensity for the Inactivated Activated Non expressing cell types 25 120 11 80 12 50 Non xpressing 0 1 2 3 inact PHOSPHORYLATION Act Statistics EEU Iis i A Statistics float Mean PI3K Total intercity 0f Cells PISK Total intensity 0fCels Inactivated UL 69 40 8 16 694 Inactivated UL 69 40 8 16 694 Select plot to adjust marker gate Activated UR 41 80 23 52 48 4 FOPULATION PROFILE di EXPRESSION iva 80 96 6 75 j 2 Non expressing Li LLI fa a Figures A and B Jurkat cells were exposed to 1uM wortmannin for 60 minutes at 37 C to inhibit the Akt signaling cascade response fixed permeabilized and then stained with both anti phospho Akt Ser473 and anti Akt PKB antibodies in multiplex Samples were acquired using the Muse Cell Analyzer and statistical results are shown above Figure A shows the results summary while Figure B shows results displayed by both dot plot and bar graph data The statistics captured in this assay show the relative percentages for each population as it is calculated
11. adrant marker is set a v anacucl statistics of the cellular popi desired location 4 Back Return to Results 12 13 If no adjustments are needed select Next Run and repeat steps 9 through 12 for the remaining samples NOTE During the run a message may appear prompting you to load a tube of DI water for a Quick Clean Load the water then select Clean to perform the Quick Clean Select Next to continue with the run The frequency of Quick Cleans was set by your system administrator Your administrator may also have chosen to allow you to skip the Quick Clean when the prompt appears You can choose to perform additional Quick Cleans at any time during a run by selecting Clean in the title bar then Quick Clean from the menu 14 When you have acquired the last sample select Finish 15 Optional Select Options in the title bar to rename the data set export the data set save the current instrument settings or view the event log Refer to the Muse Cell Analyzer User s Guide for more information 13 Quick Clean Load sample tube containing DI Water Amm nj ect Clear J J pi Results The software performs calculations and displays the data in two plots e A dot plot displaying cells which are inactivated non expressing and activated on a bivariate plot indicating Akt expression and phosphorylation or dephosphorylation for a given testing sample e A bar graph illustration of the same dat
12. and cell proliferation The phosphorylation of Akt is the central node for this signaling cascade hence providing both total and phospho Akt antibodies for accurate measurement of PISK Akt signaling Gluconeogenesis Summary of Protocol Prepare cell cultures for experimentation dd 10 uL of the antibody treated or untreated cocktail to 90 uL of 1X Resuspend in 200 uL 1X ssay Buffer per tube test Assay Buffer and Centrifuge cells at 300 x g for 5 min and low to incubate for 30 min acquire samples on wash once with 1X PBS at room temp dark Muse Cell Analyzer l abe Fix cells in Fixation Buffer for 5 min on ice followed by a washing step rT in c Permeabilize cells with Permeabilization Buffer for 5 min on ice followed by a AS J washing step i Centrifuge cells at Add 200 000 cells to each tube 300 x g for 5 min treated or untreated and wash with 1X Assay Buffer Kit Components e 20X Anti phospho Akt Ser473 Alexa Fluor amp 555 Part No CS208206 One vial containing 250 uL e 20X Anti Akt PKB PECy5 Part No CS208207 One vial containing 300 uL e 5X Assay Buffer Part No CS202124 One bottle containing 55 mL e Fixation Buffer Part No CS202122 One bottle containing 13 mL e Permeabilization Buffer Part No CS202125 One bottle containing 13 mL Materials Not Supplied Test tubes for sample preparation and storage Tissue culture reagents i e HBSS PBS w o Ca or Mg
13. by both dot plot and bar graph data The statistics captured in this assay show the relative percentages for each population as it is calculated within the total cell population Cells which express Akt can be seen by the data on the top two quadrants of the dot plot inactivated and activated representing about 94 996 of the total cell population But of this cell population 73 8 is activated upon treatment confirming the activation of the Akt signaling pathway in stimulated NIH3TS cells By presentation of both datasets one can now determine the total phospho ratio within their testing samples 15 Technical Tips 1 For cellular staining and analysis to be most effective make sure that test cells have good viability prior to use For certain cell cultures cell pellets may become hazy or transparent following the fixation step making it difficult to see If sampling a small collection of cells for flow analysis it is recommended that all steps be performed in a smaller collection tube e g centrifuge tube Do not mix or interchange reagents from various kit lots Mix each cell sample thoroughly on a mixer before acquiring samples for consistent and accurate results However avoid vigorous mixing which can cause cellular breakdown and splashing resulting in volume loss and erroneous results The default number of events to acquire is 1000 events You may select a different number however your statistical error w
14. cell dislodging buffers etc Pipettes with corresponding tips capable of accurately measuring 10 1000 uL Tabletop centrifuge capable of achieving 300 x g Mechanical vortex Deionized Water for buffer dilution Cells of interest in suspension e g Jurkat NIH3TS etc NS XO ev SS Jue a Microcentrifuge tubes with screw caps 1 5 mL VWR Catalog No 16466 030 or equivalent 9 Muse Cell Analyzer 10 Muse System Check Kit Catalog No MCH100101 11 Guava ICF Instrument Cleaning Fluid Catalog No 4200 0140 optional Precautions e The instructions provided have been designed to optimize the kit s performance Deviation from the kit s instructions may result in suboptimal performance and may produce inaccurate data e Some assay components included in the kit may be harmful Please refer to the MSDS sheet for specific information on hazardous materials MSDS forms can be found on the web page or by contacting Millipore technical services e During storage and shipment the directly conjugated antibodies may condense within the vial For maximum recovery of the product centrifuge original vial prior to removing cap e The conjugated antibody is light sensitive and must be stored in the dark at 2 8C e Do not use reagents beyond the expiration date of the kit Storage All reagents must be stored at 2 8 C All kit components are stable up to four 4 months from date of receipt if stored and handled correctly Pl
15. ease avoid repeated changes in temperature as this will affect the integrity of the product Before You Begin It is highly recommended that you run the cell samples shortly after completing the sample preparation While some cell types have been shown to yield stable results for up to 24 hours after cell fixation permeabilization antibody staining if properly stored the stability of individual cell types may vary Time considerations When dealing with phospho specific activation detection fixation of cell samples after cell treatment s is critical to capture the phosphorylation activation event Some activation state cell signaling responses are transient and may be lost if cell cultures are not fixed immediately following treatment Cell fixation permeabilization and staining will take approximately 50 minutes Acquiring data on your Muse Cell Analyzer takes less than 3 minutes per sample depending on the cell concentration and desired number of events to acquire Always perform a System Check prior to performing the assay For details refer to the Muse Cell Analyzer User s Guide Preparation of Reagents 1 Assay Buffer Assay Buffer is supplied at 5X concentration and should be diluted to 1X with deionized water prior to use Prepared 1X Assay Buffer is stable up to one year Store at 2 8 C 2 Antibody Working Cocktail Solution The kit contains two 2 antibodies which can be used in multiplex Prior to antibody staining of
16. ently A low signal may indicate that the cells need to be stained at a higher volume Verify that the System Check procedure was performed and the results passed Variability in day to day If the results are inconsistent check that the samples were well mixed experiments prior to acquisition Cells may quickly settle in your samples and your results will be inaccurate unless the cells are mixed just prior to acquisition Monitor experimental cell cultures to ensure that cell viability and cell numbers being analyzed are consistent Any drop in cell numbers or viability can influence experimental results If there appears to be day to day variation of the staining pattern ensure the Muse Cell Analyzer is working properly Check the Muse System Check log to ensure day to day instrument variation is low acquisition 17 References 1 Alessi D R et al 1998 3 Phosphoinositide dependent protein kinase 1 PDK1 phosphorylates and activates the p70 S6 kinase in vivo and in vitro Curr Biol 8 2 69 81 2 Cohen P et al 1997 PDK1 one of the missing links in insulin signal transduction FEBS Lett 410 1 3 10 3 Alessi D R et al 1997 3 Phosphoinositide dependent protein kinase 1 PDK1 structural and functional homology with the Drosophila DSTPK61 kinase Curr Biol 7 10 776 89 4 James S R et al 1996 Specific binding of the Akt 1 protein kinase to phosphatidylinositol 3 4 5 triphosphate withou
17. ill increase as you decrease the number of acquisition events If results deviate from expected values prepare a freshly stained sample and reacquire the data Periodically run Quick Clean using a tube of DI water after every 20 sample acquisitions to prevent a buildup from cellular debris in the system If your samples contain significant amounts of cellular debris run the Quick Clean cycle more often to prevent clogs or blockage If you are acquiring data from a sample but the progress bar is not moving there is probably either insufficient volume to continue to acquire the sample or a blockage of the flow system First check to ensure that there is at least 100 uL of sample in the tube If not add additional buffer to bring the volume up to 100 uL or proceed to the next sample If the sample volume is greater than 100 uL then the lack of events is probably due to a clog A clog or blockage can be caused by cell aggregates cell debris bleach crystals or other particulates Perform a backflush to flush out the clog into a tube containing 20 bleach Then run Quick Clean to remove bleach residue If this procedure does not alleviate the problem refer to the Muse Cell Analyzer User s Guide for additional troubleshooting tips or contact Millipore Technical Support for help The Muse PISK Activation Dual Detection Kit works best with samples in single cell suspension Cell aggregates may clog or be excluded from the flow ce
18. ion relative to the total Akt expression in any given cell population By doing such the levels of both the total and phosphorylated protein can be measured simultaneously in the same cell resulting in a normalized and accurate measurement of PI3K activation after stimulation Moreover simultaneous measurement of both total and phospho Akt confirms target specificity of the phosphorylation event Together a total and phospho antibody duo performed in multiplex provides an enhanced and more reliable detection of the phospho total ratio within a mixed population The antibody pair provided in the kit has been carefully titrated to ensure the ability to measure total and phospho Akt simultaneously on the same protein for accurate determination of protein level and 1 activation Sufficient reagents are provided to perform 50 tests Detailed assay instructions are included to assist in analysis and to ensure the correct cell concentration is obtained during acquisition of sample data For Research Use Only Not for use in diagnostic procedures I Integrin Torc 1 _ Cell Growth ra Protein Synthesis Cell e eos Morphology e e Permeability Torc 2 Proliferation Differentiation p Catenin J PI3K Akt Signaling Pathway As indicated in the diagram above the PISK Akt signaling pathway has many branching points which regulate a large number of cellular processes such as cell survival cell growth
19. lect Next otherwise select Back lf settings are correct select Next otherwise select Back 10 9 Enter the sample ID by touching the field then using the keypad to input the ID Touch Done when you re finished entering the ID If necessary change the Events to Acquire by touching the field then selecting the value from the pop up menu Select Next Home Assay Settings Run Sample Info Sample 001 Sample ID Sample 001 2 Events to Acquire 1000 Events ww 10 Follow the onscreen instructions and mix the first sample Load the sample on the instrument loading arm Select Run to acquire the sample Run sample During acquisition live statistical values are generated and plotted onto a bar graph Measurements POPULATION PROFILE oo include inactivated activated and iid Sic non expressing cells Mix and Load Raise Sample Sample Loader Select Run Example shown Jurkat cells treated with Wortmannin to cause deactivation of Akt signaling Neg PI3K EXPRESSION Pos 0 1 2 3 4 Inact PHOSPHORYLATION Act Next Stop amp discard run Skip rest of events 11 11 When acquisition is complete the results are displayed If desired select Plots to display a dot plot and a bar graph for the sample Home Assay Settings Run Home Assay Settings E ii Options Clean Options Clean Lili 4 004 jurkat t mix 004 jurkat t mix
20. ll affecting the accuracy of the results If you wish to use the Muse PISK Activation Dual Detection Kit with a clumpy cell line it is recommended to order Muse M Cell Dispersal Reagent Catalog No MCH100107 to disaggregate the cells Contact customer service or visit our website at www millipore com muse for detailed information on the Muse M Cell Dispersal Reagent and assay method For more troubleshooting tips refer to the Muse Cell Analyzer User s Guide For more information contact the Millipore office nearest you In the USA call 1 800 MILLIPORE 1 800 645 5476 Outside the USA visit our website at www millipore com offices for up to date worldwide contact information You can also view the tech service page on our website at www millipore com techservice 16 Troubleshooting Potential Problem Experimental Suggestions Acquisition taking Ensure that the System Check procedure was run and passed If the longer than expected progress bar stops during acquisition the fluid system may be clogged Run or a Quick Clean procedure to clean the capillary It can be performed during progress bar stops or after an assay during acquisition Instrument clogging If the instrument is clogged run a Quick Clean procedure to clean the capillary It can be performed at anytime during an assay between samples No detectable oince phospho specific activation can be very quick transient responses in phosphorylation order to capture
21. nalyzer Run a System Check prior to performing the assay For information on Muse System Check refer to the Muse Cell Analyzer User s Guide 1 Select MAPK Activation from the main menu All Assay Search MitoPotential MultiCaspase a PI3K Activation i STAT 3 Activation 2 Select Run Assay Home Assay Settings Run Results PISK Activation Run Assay View Results 3 Adjust the instrument settings e Load the sample for adjusting the settings and select Run Note Perform the adjust settings step using a Silis i Retrieve Settings negative control e g total antibody then verify the settings using a positive control Mix and Load Raise e Or to retrieve previously saved instrument Sample Sample Loader Select Run settings select Retrieve Settings For more information on retrieving settings see the Muse Cell Analyzer User s Guide Cancel Cancel amp Eject 4 Fine tune the settings for the PISK EXPRESSION and CELL SIZE INDEX plot if necessary e Adjust the CELL SIZE INDEX slider accordingly to capture the cell population of interest see on screen instruction for example e Drag the threshold left or right to exclude cell debris Drag to make large changes Touch the arrow buttons located below the plot to make small changes The arrow buttons appear after you touch the threshold function NOTE If the acquisition times out after two minu
22. nt markers to place the cell population s immediately to the left of the marker see diagram below Example 1 However in certain cases the activation of Akt can be constitutively activated e g human Jurkat cells And if evaluating PI3K inhibitors such as Wortmannin LY294002 or U0126 adjust the quadrant markers to place the cell population if activated immediately to the right of the markers instead see diagram Example 2 Addition of such compounds will cause a left population shift or deactivation of phospho specific Akt signaling Adjust the quadrant markers You can move the marker intersection in any direction as well as adjust the angle of each line To move the markers as they are touch the open circle at the intersection and drag the markers to make large changes or touch the arrow buttons below the plot to make small changes A To adjust the angle of either line touch the solid circle and drag in an arc or touch the arrow buttons below the plot B and C Example 1 Mouse NIH3T3 cells Inactivated A Moving the quadrant marker B Adjusting the X axis C Adjusting the Y axis POPULATION PROFILE PI3K EXPRESSION eg N G2 Q PI3K EXPRESSION N PI3K EXPRESSION eg Non e Non e pem N o 0 1 2 3 E Inact PHOSPHORYLATION Act act PHOSPHORYLATION M act PHOSPHORYLATION M ps a Li POPULATION PROFILE 1 To set POPULATION PROFILE 1 To set 3
23. t subsequent activation Biochem J 315 Pt3 709 13 5 Cross D A et al 1995 Inhibition of glycogen synthase kinase 3 by insulin mediated by protein kinase B Nature 3 8 6559 785 9 Related Products Muse H2A X Activation Dual Detection Kit Catalog No MCH200101 Muse EGFR RTK Activation Dual Detection Kit Catalog No MCH200102 Muse MAPK Activation Dual Detection Kit Catalog No MCH200104 Muse Bcl 2 Activation Dual Detection Kit Catalog No MCH2001 05 Muse System Check Kit Catalog No MCH100101 Muse Count amp Viability Kit 100T Catalog No MCH100102 Muse Count amp Viability Kit 600T Catalog No MCH6001 03 Muse Count amp Viability 200X Catalog No MCH100104 9 Muse Annexin V amp Dead Cell Kit Catalog No MCH100105 10 Muse Cell Cycle Kit Catalog No MCH100106 11 Muse Caspase 3 7 Kit Catalog No MCH100108 12 Muse MultiCaspase Kit Catalog No MCH100109 13 Muse Mitopotential Kit Catalog No MCH1001 10 14 Muse Cell Dispersal Reagent Catalog No MCH100107 pucr one b ge ine cae cet 18 Warranty EMD Millipore Corporation EMD Millipore warrants its products will meet their applicable published specifications when used in accordance with their applicable instructions for a period of one year from shipment of the products EMD MILLIPORE MAKES NO OTHER WARRANTY EXPRESSED OR IMPLIED THERE IS NO WARRANTY OF MERCHANT ABILITY OR FITNESS FOR A PART
24. t to have a dominant role in growth survival embryonic development and adipocyte differentiation Akt2 is correlated with the regulation of glucose homeostasis and is the predominant isoform expressed in insulin responsive tissues Akt3 is abundant in brain tissue Each Akt isoform is composed of three functionally distinct regions an N terminal Pleckstrin Homology PH domain that provides a lipid binding module a central catalytic domain containing Thr308 and a C terminal hydrophobic motif containing Ser473 The activation of AKT is dependent on a dual regulatory mechanism that requires both its translocation to the plasma membrane and dual phosphorylation on Thr308 and Ser473 by PDK1 and the TORC2 complex respectively All Muse Dual Detection kits are optimized on the Muse Cell Analyzer Both antibodies provided in the kit are carefully titrated and optimized together to ensure maximal performance when run in multiplex alleviating the need for any additional optimization This kit contains optimized fixation permeabilization and assay buffers to provide researchers with a complete solution for PI3K signaling analysis Test Principle The Muse PI3K Activation Dual Detection Kit includes two directly conjugated antibodies a phospho specific anti phospho Akt Ser473 Alexa Fluor amp 555 and an anti Akt PECy5 conjugated antibody to measure total levels of Akt This two color kit is designed to measure the extent of Akt phosphorylat
25. tes you can select Abort to restart the adjust settings step or Next to accept the settings and continue to the next step Settings Step 1 of 2 Pos Pos Capture cell population of interest by adjusting the CELL SIZE INDEX slider bar on the X axis PI3K EXPRESSION PI3K EXPRESSION Touch threshold to activate and adjust left right to exclude cell debris 4 Neg Neg 1 Use slider to place cells in optimized region PISK EXPRESSION PI3K EXPRESSION 2 Drag threshold to exclude debris CELL SIZE INDEX CELL SIZE INDEX Abort Discard Changes Abort Next Discard Changes Set Population Profile CELL SIZE INDEX Next Set Population Profile Fine tune threshold adjustments by using the arrows below 5 Select Next when you have completed the adjustments 6 Fine tune the settings for the PISK EXPRESSION vs PHOSPHORYLATION plot if necessary SETTING THE GATE To set the quadrant marker properly prepare a cell sample containing only the total antibody e g 5 uL of anti Akt PKB PECy5 antibody 90 uL 1X Assay Buffer in a cell suspension Adjust the slider bars on the X and Y axis to place all populations Non expressing Inactivated and Activated on scale If the cell sample is not treated e g activated a great majority of the cell population will fall either in the Inactivated upper left or Non expressing quadrants lower left Adjust the quadra
26. umption or application to humans or animals c 2009 2012 Merck KGaA Darmstadt All rights reserved No part of these works may be reproduced in any form without permission in writing 19 Cat No MCH200103MAN October 2012 Revision A
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