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1. USB 2 0 Cable Power Supply 2009 Nexc elom Bioscience LLC Vision Manual 19 Getting Started Initial Configuration Setting Background Image Before using Cellometer Vision a background image image of the system itself without a counting chamber must be obtained to nomalize the instrument Unlessthe instrument is moved there is generally no need to take a background image again Step 1 Ensure Cellometer Vision is connected to the laptop controller and tum on both the Cellometer and the Laptop Step 2 Double Click on the Cellometer Vision icon on the Laptop desktop to launch the software Step 3 The first time the software launches you may see this message Click OK to continue NOTE You will not see this message when using a laptop supplied directly by Nexcelom Bioscience orAuthorized Distributor Cellometer Error Can nat load background image specified as C Program FilesiWNexcelomi Background bmp Please take a background image after the program starts Step 4 Click Preview Brightfield Image and verify a gray uniform background image Preview Bnghtfield Image Step 5 From the Menu bar select Options Take Background image to begin image acquisition NOTE This procedure can take several secondssince it automatically adjuststhe exposure to obtain the optimal background image 20 2009 Nexcelom Bioscience LLC Using Vision forthe First Time Using Vision for the First Time
2. W Cellometer Vision Trio 5 3 File Assay Type Options Help Cell Count E Ji F1 Count iv Tint F SETUP View Image Assay Assay 05_YFP Tran A Cell Type Cell Line No decluster FA Imaging Mode BA Image F1 Image F2 Image Brightfield and Fluorescence SAMPLE Sample ID ES Cell YFP Counted Dilution 1 Count In View Preview Brghtield Image Manual Adjust Exposure ms D En 2000 0 meee Step 5 Adjust Focus if necessary Step 6 Click Preview F1 Image Fluorescence IN Cellometer Vision Trio 5 l SI File Assay Type Options Help Cell Count 0 F1 Count H Sen EST PZ Cell Type Cell Line No decluster 4 FZ Imaging Mode E f EF Image Brightfield and Fluorescence z F1 Image F2lmage SAMPLE Sample ID SSC mc ES Cell_ YFP lll Counted Dilution Count In View Preview Briahtield Image Manual Adjust Exposure ms 2000 0 mec Step 7 Click Count 2009 Nexc elom Bioscience LLC Vision Manual 53 Tutorials Step 8 Review Counting Results and display data asneeded Counting Results Sample ES Cell YFP Dilution 1 0 Assay Assay 05 YFP Transfection Rate Description Cell line transfection rate measurement using YFP Cell Cell Line Mo decuster Assay 05 Description Cell line cells not dumpy Bright Field Fluorescence a 240 b h Show Size Distribution T Mean S
3. Counted cellsindicated by the green outline 2009 Nexcelom Bioscience LLC C Fluorescence cell image D Counted fluorescence positive cells indicated by the green outline The red outline indicated cells that are fluorescence negative Assay 03 GFP Transfection Rate 2009 Nexc elom Bioscience LLC Vision Manual 43 Tutorials Step 10 To adjust fluorescence threshold to determine which cells are counted asfluorescent positive or negative adjust Cell Type Parameters by clicking on the pencil icon next to Cell Type Fluorescence Threshold is user defined to identify the brightness at which a cell is defined as positive Cell Type Name Cell Line_Assay 03 a ee Qe iue Cell is Locked fram Editing Detailed Description Bright Field BR Fluorescence FL Description Minimum Maximum Cell Diameter 4 micron 33 micron Iv Normalize Intensity for Cell Size Roundness 0 00 default 0 10 range 0 1 0 1 0 for perfect circle Remove stray DNA Combine with BR Images Fluorescence Threshold Parameters Auto Threshold Fluorescent Count range 0 100 of brightest cell 10 0 Lower values count dimmer cells Ce Manual Threshold Fluorescent en Countrange 0 100 of image max Lower values count dimmer cells Decluster Hug default 0 05 range 0 1 0 Th Factor WE lower value for better decluster GFP fluorescence image Threshold 375 Threshold 2075 F1 positive rate 58
4. Note Assay parameters for assays that are included in the software are locked and can not be edited To customize this assay to suit your particularapplication please see Modify Default Assay Parameters for instructions on how to modify and save asa new assay Step 1 Mix cell samples with Caiceim AM and incubate according to manufacturer s instructions Step 2 Load 20uL cell sample in one side of the cell counting chamber and insert into instrument Step 3 Select Assay 09 Calcein AM Macrophage from the Assay SETUP drop down menu Imaging Mode Bnghtfield and Fluorescence Image mode Brightfield and Fluorescence Click on the yellow pencil on the right side of Assay selection to view Assay Type Assay Name Assay 09 Calcein AM Macrophage e Assay is lacked from editing Save as New Assay Type Special Cells Description Viable cell concentration using Calcein AM Imaging Mode Brightfield BR amp Fluorescence F 1 Trypan Blue Viability Use BR Cell for Fi Count F1 Image Cell Type Macrophage CA Assay 09 Description Edit Fluorophore Calcein 101 E Fluorescent Exp 1000 0 mser Filter Set Step 4 Click Preview Bnghtfield Image Step 5 Adjust focus if necessary Step 6 Click Preview F1 Image 72 2009 Nexcelom Bioscience LLC Sep 7 Click Count Step 8 Review Counting Results and display data asneeded Counting Results Sample Macrophage CA Dilution 2 0 Assay Assay
5. The following isa procedure to modify an existing assay type to suit specific experiments Step 1 Select Assay 03 GFP Transfection Rate from Assay SETUP drop down menu Cell Type Cell Line Assay 03 Imaging Mode Bnghtfield and Fluorescence Step 2 Edit Assay Type by click on the yellow pencil tool Assay Name Assay 03 GFP Transfection Rate Lock Assay from future editing i Save as New Assay Type Special Cells Description Cell line transfection rate measurement using GFP Imaging Mode Brightfield BR amp Fluorescence F 1 Trypan Blue Viability Iw Use BR Cell for Fi Count El Image Cell Type cel Line Assay 03 Description EE Fluorophore GFP Ex 475nm 101 Fluorescent Exp 000 0 mser Filter Set Frotocol Notes Browse Show Report Format Show Print Cancel E a Check Save as New Assay Type b Input new or modified name in Assay Name Save will tum on afterthe assay name is changed c Modify cell type if necessary Method 1 drop down from F1 Image Cell Type to find anothercell type Method 2 Edit to change curent cell type parameters 90 2009 Nexcelom Bioscience LLC Modify Default Assay Parameters Cell Type Name Cell Line_Assay 03 Save as New Cell Type Tell E s Locked from Editing Detailed Description Bright Field BR Fluorescence FL Minimum Maximum micron 35 micron Roundness 0 10 default 0 10 range 0 1 0 1 0
6. 14 11 13 15 40 14 6 18 15 11 55 16 4 60 17 12 86 18 14 26 19 16 08 20 10 79 Cell Size Microns 21 13 81 3 pen Size File pen Intensity File Copy to Clipboard Cancel O 2009 Nexc elom Bioscience LLC Vision Manual 55 Tutorials k Sample Adjustment Calculator Sample Adjustment x Sample Adjustment Measured Concentration cells ml 3 42e 005 Measured Concentration cells ml 3 42e 006 Original 5 ample Volume ml 1 0 Original ample Volume ml 1 0 Total Cell Humber in S ample 3 42e 006 Total Cell Number in Sample 3 42e 005 w Target Concentration cells ml 1 00e 006 Target Concentration cells ml Target Number of Cells iw Target Number of Cells 100000 PIN Sample Adjustment Sample Adjustment Spin down and add diluent amount 0 34 ml Take 292 20 ul of sample Good for 3 aliquots v Print with report Iw Print with repart Calculator 1 input target cell Calculator 2 input target number of concentration and calculate needed Amount of sample to add and adjustment the number of aliquots will be calculated 56 2009 Nexcelom Bioscience LLC Assay 05 YFP Transfection Rate Step 9 Review Images on screen r View Image DN Zoom In El Image F1 Image F2 Image v Combined iv Counted A one of the fourcaptured images 1 2 3 or 4 one quadrant of a selected image FL fluorescence 1 image view F2 fluorescence 2 image view Combined check
7. 7 Click Count 46 2009 Nexcelom Bioscience LLC Assay 04 RFP Transfection Rate Step 8 Review Counting Results and display data asneeded Counting Results Sample SF9 RFP Dilution 1 0 Assay Assay 04 RFP Transfection Rate Description Cell line transfection rate measurement using RFP Cell Cell Line Assay 04 Description Bright Field Fluorescence a 1292 524 h Show Size Distribution Mean Size C 13 0 d 20 4 Intensity Distribution BR Size j Size vs Intensity Concentration 1 87 x10 P f 9 09 x10 7 j k Sample Adjustment EE Set Data File BR Total Count lY save to Data File M ven Data File Count brightfield total number of cells counted in the brightfield image Count Fluorescence number of fluorescence positive cells counted Mean Size brightfield mean diameter of cells measured in brightfield Mean Size Fluorence mean diameter of fluorescence postive cells Concentration Bright Field total cell concentration mL Concentration Fluorescence fluorescence cell concentration mL g Fl Count BR Total Count transfection rate o Be ae ds h Show Size Distribution displays cell size histogram generated using brightfield image i Intensity Distribution displays fluorescence cell intensity histogram j Size vs Intensity displays fluorescence intensity vs cell size scatterplot k Sample Adjustment calculates adjustment for target
8. 85 Tutorials Step 6 Review cell images Preview H1 Freview F1 Es posure nri 1TO0 of Range 1000 0 msec Preview F2 Exposure ms m 2000 0 mec Click BT Preview BI will display brightfield image of F1 channel Adjust Focus if necessary Stop Review Click Preview EU Fluorescence 100 of Range indicates adequate exposure time for Fl channel g Click Preview F2 Fluorescence hm Oo CO O Cellometer Warning Fluorescent intensity is only 14 of the full range Consider increasing the exposure time by a min of 6 5x h If exposure time is inadequate increase exposure time by using the pencil tool next to the Assay Set up drop down menu 86 2009 Nexcelom Bioscience LLC Assay 12 Splenocyte Concentration and Viability Using AO EB Step 7 Review Counting Results and display data asneeded Counting Results Sample Splenocyte AQEB 2 Dilution 2 0 Assay Assay 12 AOEB Splenocyte new Description 5plenocyte stained with AOEB mixture to measure live dead cells F1 Cell Splenocyte Assay 12 F2 Cell Splenocyte_Assay 12 F1 Description F2 Description Fluorescent 1 Fluorescent 2 Count 852 b 1160 nu 2 39 x10 6 d 3 25 x10 b Fi Count F1 F2 Count f Save to Data File view Data File a Count Fluorescent 1 indicates total number of live cells b Count Fluorescent 2 indicates total number of dead cells c Concentration Fluorescen
9. PBMCsaftercounting Green PBMCs outline indicates counted cells 66 2009 Nexcelom Bioscience LLC Assay 08 Cell Viability Using Propidium lodide Staining Assay 08 Cell Viability Using Propidium lodide Staining Propidium iodide is routinely used to determine cell viability Pl isa fluorescent stain that only penetrates dead cells and emits in the red range live cells are unaffected by PI After taking both bnghtfield and red fluorescent images of a PI stained sample all cells from brightfield channel and dead cells from red fluorescent channel are counted to determine total and dead cell concentrations and compute the percent viability Note Assay parameters for assays that are included in the software are locked and can not be edited To customize this assay to suit your particularapplication please see Modify Default Assay Parameters for instructions on how to modify and save aSa new assay Step 1 Mix cell samples with Pl and incubate using the same method as when manually counting Step 2 Load 20uL stained cell sample in one side of the cell counting chamber and insert into instrument Step 3 Select Assay 08 PI Viability J urkat from the Assay SETUP drop down menu Cell Type Uurkat Pl Ass Imaging Mode Bnghtfield and Fluorescence Imaging mode Brightfield and Fluorescence Click on the yellow pencil on the right side of Assay selection to view Assay Type Click Show Report Format to display
10. Serial amp Label 18 2009 Nexcelom Bioscience LLC Setting up Cellometer Vision Setting up Cellometer Vision All Nexcelom products undergo a rigorous quality inspection pnorto shipment and all reasonable precautions are taken in preparing them for shipment to assure safe delivery The instrument should be unpacked and inspected for mechanical damage upon receipt Mechanical inspection involves checking for signs of physical damage such as broken knobs scratches dents etc If damage isapparent orany components are missing please immediately contact Nexcelom 1 978 327 5340 or support nexcelom com or your local dealer After unpacking the instrument and laptop plug the Cellometer Power Cable into the back of the instrument and connect the instrument to the laptop using the enclosed USB 2 0 cable If you use a computer thatis not supplied directly from Nexcelom Bioscience or Authorized Distributor the software must be installed BEFORE the USB 2 0 cable is connected If you are using a computer supplied directly by Nexc elom Bioscience orauthorized distributor connect the instrument to the computer with the enclosed USB 2 0 cable The laptop is pre configured with Cellometer Vision software and Microsoft Excel North America only No additional setup orconfiguration is required If software re installation is required please contact Nexcelom Technical Support or yourlocal dealer Connecting Cellometer Vision to Laptop
11. Size for Histogram Separate Fluorescent Positve Negative Cells for Cell Size Histogram lw Show Percent F 1 BR W Show Sample Adjustment lf Show Cell Size Distribution Ww Show Cell Intensity Distribution w Show Cell Size Intensity Distribution Iw Show Data File Buttons Print E Cancel i Show Report Format select to display Counting Results display option buttons that appearon the nght hand side of the Counting Results box The following options allow you to customize which results are displayed in the Counting Results dialog box Show Cell Count Check to display cell counts Show Trypan Blue Dead Cell Count Check to display dead cell counts Show Trypan Blue Viability Check to display the viability Show Cell Mean Diameter Check to display the mean diameter of counted cells Show Trypan Blue Dead Cell Mean Diameter Check to display the mean diameter of trypan blue stained dead cells Show Dilution Check to display the dilution factor entered in the main window Show Concentration Check to display the cell concentration For Fluorescent Positive Cells Use Brightfield Cell Size for Histogram Check to use the brightfield cell size when generating the cell size histogram for fluorescent cells Separate Fluorescent Positive Negative Cells for Cell Size Histogram Check to generate a cell size histogram with fluorescent positive cells shown as green bars and fluorescent negative cellsasred bars Show Percent F1 BR Indicateshow
12. and Fl images Counted check to see counted cells outlined When both fluorescence channels are used check the Combined box to display an overlay of F1 and F2 captured images A Cell image before counting B Cell image after counting Green outline indicateslive cellsand red outline indicates trypan blue positive dead cells 2009 Nexc elom Bioscience LLC Assay 03 GFP Transfection Rate Assay 03 GF Transfection Rate Transfection efficiency is often determined by using a GFP marker either on the vector of interest or co transfected with the vector Aftertaking brightfield and fluorescent images of the transfected sample all cellsare counted in brightfield and fluorescent images Transfection efficiency is computed by dividing the number of fluorescent cells by the total number of cells Note Assay parameters for assays that are included in the software are locked and can not be edited To customize this assay to suit your particularapplication please see Modify Default Assay Parameters for instructions on how to modify and save aSa new assay Step 1 Trypsinize attached cells Step 2 Load 20uL in one side of the cell counting chamber and insert into instrument Step 3 Select Assay 03 GFP Transfection Rate from Assay SETUP drop down menu Imaging Mode Bnghtfield and Fluorescence Cell Type HCT is displayed Imaging Mode Brightfield and Fluorescence The brightfield imaging mode is used to measure total
13. are used check the Combined box to display an overlay of F1 and F2 captured images a E m ranas eet di pd A ae E SN wer CE e Self ul yf LI m A g F i A Brig htfield image showssample with crowded red blood cells 2009 Nexcelom Bioscience LLC Vision Manual 61 Tutorials B Fluorescent cell image before counting C Fluorescent cell image after counting outline Green indicates counted white blood cells 62 2009 Nexcelom Bioscience LLC Assay 07 PBMC Concentration Using Acridine Orange Staining Assay 07 PBMC Concentration Using Acridine Orange Staining PBMC preparations generally contain some RBCs making it difficult to accurately count only the PBMCs RBCscan be eliminated to improve counting by using a lysis procedure However PBMCscan be selectively counted without lysis of RBCs by staining the PBMC preparation with Acridine orange a nuclear stain that emits in the green range Aftertaking green fluorescent images all fluorescent cells are counted and their concentration in the PBMC preparation is determined Brightfield images of the sample can be taken but are not used for PBMC counting Note Assay parameters for assays that are included in the software are locked and can not be edited To customize this assay to suit your particularapplication please see Modify Default Assay Parameters for instructions on how to modify and save asa new assay Step 1 Mix ce
14. concentration ortarget number of cells L Set Data File creates a data file to store cell counting table m Save to Data File records counting results in the cell counting table n View Data File displa ys currently selected cell counting table o Export stores all raw data assay parameters and graphs in an Excel file p Print sendsthe cument counting resultsto a printer q Done closescounting results window Note After clicking Done the Counting Results box can be redisplayed at any time by clicking on the View Report of Counting Results button O 2009 Nexcelom Bioscience LLC Vision Manual 47 Tutorials h Cell size distribution for brightfield image Cell Size Analysis Diameter File Name C Documents and Settings ll Usersk pplication Data Nexcelom_Vision5 diameters F1 txt Cell Diameters Histogram __ Diameter um 1 10 79 7 2 21 93 3 17 89 4 27 23 5 23 65 6 18 92 7 24 44 8 28 26 E 3 18 75 10 19 80 8 11 24 96 e 12 25 84 13 23 15 14 18 58 15 20 74 16 23 11 17 24 79 Total Cell Count 18 17 16 19 13 63 Mean Diameter 20 19 09 21 21 04 3 Open Diameter File Multiple Save As NOTE this can be instantly displayed at any time by clicking onthe ull icon i Fluorescence intensity versus number of cells Cell Intensity Analysis Intensity File Name C Documents and Settings ll Users pplication Data Nexcelom_Vision5 intensity BRFL txt Cell Inte
15. for perfect circle 0 40 Cell Diameter Contrast default 0 40 range 0 0 8 Enhancement high value for light cells Decluster Parameters NoDeduster Decluster Edoe Factor default 0 5 range 0 1 0 higher value for more edge enhancement Decluster Th 1 0 default 1 0 range 0 1 0 Factor higher value for more sensitivity Background 48 default 1 0 range 0 1 0 Adjustment lower value to pick up dim cells Trypan Blue Viability Parameters Minimum Maximum Dead Cell 3 i micron 5n Diameter ea io default 1 0 range 0 6 0 EEN higher value to pick up more dead cells default 150 range 100 255 higher value for non uniform dead cells Uniformity M Very Dim Dead Cells Contrast o9 default 0 40 range 0 0 8 Enhancement high value for light cells Cancel Check Save as New Cell Type Change Cell Type Name Input modification to the cell type parameters Save d Change Fluorescence exposure time if needed 2009 Nexcelom Bioscience LLC Vision Manual 91 Tutorials 92 2009 Nexcelom Bioscience LLC Operation Reference Operation Reference 94 2009 Nexcelom Bioscience LLC Software Operation Overview Software Operation Overview The following section descnbes each function available in the software main menu File Menu Assay Type Options H Load Image for Display Load Image and Count New Data File Select Data File Vi
16. iscalculated Ex To show percentage of Fluorescent Positive cells choose F1 BR 100 To show percentage of Fluorescent Negative cells choose BR F1 BR 100 Note Additional calculations appear in dual fluorescence modes We Show Percent F 1 BR 1 BR 10095 FI BR 100 Show Sample Adjustment Check to show the Sample Adjustment button Show Cell Size Distribution Check to show the Show Size Distribution button Show Cell Intensity Distribution Check to show the Intensity Distribution button Show Cell Size Intensity Distribution Check to show the Size vs Intensity button Show Data File Buttons Check to show the Set Data File Save to Data File and View Data File buttons Print print out selected Cell Type parameters Cancel close Assay Type without saving changes Save saves changesto options 2009 Nexcelom Bioscience LLC Vision Manual 99 Operation Reference Assay Type gt Delete Assay Type Delete selected Assay from Setup Assay drop down list Assay Type gt Cell Type Manager Cell Type Manager Import fram Library e Nexcelom Cell Library Browse Cell Types in Cell Library Cell Types in Drop down Menu 293 Trypan Blue Adipocyte Bodipy Adipocyte Bodipy Adipocytes AC Adipocytes AC Cell Line Assay 0 Cell Line Assay 0 Cell Line Assay HO Import Cell Line Assay HO Cell Line_Assay 03 Highlighted gt gt Cell Line_Assay 03 Cell Line
17. line transfection rate measurement using GFP Imaging Mode Brightfield BR amp Fluorescence F1 Trypan Blue Viability M Use BR Cell for F1 Count F1 Image Cell Type Cell Line Assay 03 Description Edit Fluorophore GFP Ex 475 nm 101 Fluorescent Exp 2000 0 msec Filter Set Protocol Notes Browse Show Report Format Show Assay Name name of assay that appearsin the Assay drop down list Description text descriptions for your assay Save as New Assay Type check to save current Assay Type with a different name User can then edit parameters for the new Assay Type Lock Assay from future editing check to prevent future editing Parameters cannot be changed later Default assays are locked in software upon install Special Cells check box iw Special Cells Adipocyte Slide Type ICHT4 xD 100 CHT4 xD 100 CHT4 xD300 Check Adipocyte to select PD300 counting chambers with largerthic kness 2009 Nexc elom Bioscience LLC Vision Manual 97 Operation Reference are Imaging Mode Imaging Mode 21 SEGOAER EECH Try ty Iv Use BR Cell for Fi Count Fi Image Brightfield BR Only Cell Type Description Weg Brightfield BR Only usesonly brightfield to count cells and measure size Fluorescence F1 only use only fluorescence to count cells Brightfield BR and Fluorescence F1 acquire both brightfield and fluorescence images Cell counting resul
18. numberof cellsand cell size The fluorescence imaging mode is used to measure fluorescence positive cells Both Cell Type and Imaging Mode are defined in the Assay parameters and can be edited from the main screen by clicking on the pencil icon Assay Name Assay 503 GFP TransfectonRate Assay is locked from editing Save as Mew Assay Type Special Cells Description Cell line transfection rate measurement using GFP Imaging Mode Brightfield BR amp Fluorescence F1 Trypan Blue Viability FF Use BR Cell for Fi Count F i Image Cell Type Cell Line Assay 03 Description Edit Fluarophore GFP Ex 475nm 101 Fluorescent Exp D msec Eier Set Protocol Motes Browse Show Report Format y op Print Cancel E 2009 Nexc elom Bioscience LLC Vision Manual 37 Tutorials Step 4 Click Preview brightield Image I cra SETUP Vision m ji Cell Count F1 Count Tint F 9 o Assay v e 0 4 Cell Type Imaging Mode i 1 9 z BR Image Brightfield and Fluorescence i v View Image Au F1 Image F2image SAMPLE Sample ID HCT116_GFP OM BEER Count In View 262 ERES MC fre Dilution 2000 0 meee Exposure ms t 2 NN Q Step 5 Adjust Focus if necessary Step 6 Click Preview Fl Image Fluorescence ed Cell Count F1 Count Tint F View Image Assay 403 GFP tal Cell Type Cell Line_Assay 03 Le Imaging M
19. that emits in the green range and is used to stain live cells Propidium iodide isa fluorescent stain that only penetrates dead cells and emits in the red range Aftertaking both green and red fluorescent images all fluorescent cells in each channel are counted and the concentration of live green fluorescent and dead red fluorescent cellSas well as viability are determined Note Assay parameters for assays that are included in the software are locked and can not be edited To customize this assay to suit your particularapplication please see Modify Default Assay Parameters for instructions on how to modify and save aSa new assay Step 1 Mix 10 ul splenocyte sample with 10 ul AOPI mixture Step 2 Load 20 ul in one cell counting chamber and insert into instrument Step 3 Select Assay 11 AOPI Splenoc yte from the Assay SETUP drop down menu Cell Type Splenocyte Assay Imaging Mode Fluorescent 1 Fluorescent 2 Cell Type Splenocyte_Assay 11 is displayed Imaging Mode Fluorescent 1 Fluorescent 2 The fluorescent 1 is used to measure live cells while the fluorescence 2 is used to measure dead cells Both Cell Type and Imaging Mode are defined in the Assay parameters and can be shown from the main screen by clicking on the pencil icon 2009 Nexcelom Bioscience LLC Vision Manual 79 Tutorials Assay Name Assay 11 AOPI Splenocyte Assay is locked from editing Save a
20. tool next to the Assay Set up drop down menu Step 7 Review Counting Results and display data asneeded Counting Results Sample Splenocytes AOPI LiveDead 2 Dilution 1 0 Assay Assay 11_AOPI_Splenocyte Description Solenocyte stained with AOPI mixture to measure live dead cells F1 Cell Splenocyte Assay 11 F2 Cell Splenocyte Assay 11 F1 Description F2 Description Fluorescent 1 Fluorescent 2 212 SU Concentration 2 96 x10 S d 12 x10 Fi Count EE Set Data File F1 F2 Count Save to Data File Export g view Data File Print h a Count Fluorescent 1 indicates total number of live cells b Count Fluorescent 2 indicates total number of dead cells c Concentration Fluorescent 1 indicates live cell concentration mL d Concentration Fluorescent 2 indicatesdead cell concentration mL e F1 Count F1 F2Count indicates percentage of live cells to total cells f Save to Data File record counting resultsin a table g Export stores all raw data assay parameters and graphsin an Excel file h Print send the current Counting Results to a printer i Done closes counting results window Note After clicking Done the counting results box can be redisplayed at any time by clicking on the View Report of Counting Results button 2009 Nexc elom Bioscience LLC Vision Manual 81 Tutorials Step 8 Review Images on screen View Image Zoom In E Image F1 Image F2 Image Combi
21. type Bright Field Count 564 Trypan Blue 332 Dead Count f Show Size Distribution Mean Size 14 6 Viability by 70 995 Trypan Blue g Sample Adjustment Set Data File Concentration e x10 ei h Save to Data File 1 I Export View Data File d i k a Count total number of cells counted b Trypan Blue Dead Count number of trypan blue postive dead cells c Mean Size mean diameter of the live cells d Viability by Trypan Blue percentage of live cells out of total number of cells measured using trypan blue method e Concentration live cell concentration mL f Show Size Distribution display cell size histogram g Sample Adjustment launch sample adjustment calculator h Save to Data File record counting resultsin a table i Export stores all raw data assay parameters and graphsin an Excel file j Print send the current counting results to a printer k Done closes counting results window Note After clicking Done the counting results box can be redisplayed at any time by clicking on the View Report of Counting Results button 2009 Nexcelom Bioscience LLC Vision Manual 35 Tutorials Step 9 Review Images on screen Zoom In BT Image Fl Image e F2 Image v Combined Wi Counted A one of the fourcaptured images 1 2 3 or 4 one quadrant of a selected image FL fluorescence 1 image view F2 fluorescence 2 image view Combined check to display an overlay of Fl
22. 09 Calcein AM_Macrophage Description Viable cell concentration using Calcein AM Cell Macrophage CA Assay 09 Description Bright Field Count CI 420 Mean Size a i D Concentration 1 18 x1 P f Export Fluorescence 260 g Print h Done a Count Bright Field total number of cells counted in the brightfield image b Count Fluorescence number of fluorescence positive cells counted c Mean Size Bright Field mean diameter of cells measured in brightfield d Concentration Bright Field total cell concentration mL e Concentration Fluorescence fluorescence cell concentration mL f Export stores all raw data assay parametersand graphsin an Excel file g Print send current counting results to printer h Done closescounting results window Note After clicking Done the counting results box can be redisplayed atany time by clicking on the View Report of Counting Results button m e x10 Assay 09 Live Cell Concentration using Calcein AM Show Size Distribution Intensity Distribution Size vs Intensity Sample Adjustment Set Data File Save to Data File View Data File 2009 Nexcelom Bioscience LLC Vision Manual 73 Tutorials Step 9 Review Imageson screen View Image a Zoom In E Image F1 Image F2 Image Combined Counted A one of the fourcaptured images 1 2 3 o
23. 276 F1 positive rate 28 975 Increase threshold value counted only high intensity fluorescence as positive 44 2009 Nexcelom Bioscience LLC Assay 04 RFP Transfection Rate Assay 04 RFP Transfection Rate Transfection efficiency is often determined by using an RFP marker either on the vector of interest or co transfected with the vector Aftertaking brightfield and fluorescent images of the transfected sample all cellsare counted in brightfield and fluorescent images Transfection efficiency is computed by dividing the number of fluorescent cells by the total number of cells Note Assay parameters for assays that are included in the software are locked and can not be edited To customize this assay to suit your particularapplication please see Modify Default Assay Parameters for instructions on how to modify and save aSa new assay Step 1 Trypsinize attached cells Step 2 Load 20uL in one side of the cell counting chamber and insert into instrument Step 3 Select Assay 04 RFP Transfection Rate from Assay SETUP drop down menu Cell Line_Assay 04 Imaging Mode Brightfield and Fluorescence Cell Type Cell Line_Assay 04 is displayed Imaging Mode Brightfield and Fluorescence The brightfield imaging mode is used to measure total numberof cells and cell size The fluorescence imaging mode is used to measure fluorescence positive cells Both Cell Type and Imaging Mode are defined in the Assay parameters and can be
24. Amount of sample to add and adjustment the number of aliquots will be calculated 2009 Nexcelom Bioscience LLC Vision Manual 49 Tutorials Step 9 Review Imageson screen U ans sacs View Image Zoom In BT Image Fl Image e F2 Image Ed Combined A one of the fourcaptured images 1 2 3 or 4 one quadrant of a selected image FL fluorescence 1 image view F2 fluorescence 2 image view Combined check to display an overlay of Fl and Fl images Counted check to see counted cells outlined When both fluorescence channels are used check the Combined box to display an overlay of F1 and F2 captured images A Brightfield cell image B Counted cells 2009 Nexcelom Bioscience LLC Assay 04 RFP Transfection Rate C RFP positive cell image D Green outline indicates counted RFP positive c ells O 2009 Nexc elom Bioscience LLC Vision Manual 51 Tutorials Assay 05 YFP Transfection Rate Transfection efficiency is often determined by using a YFP marker eitheron the vectorof interest or co transfected with the vector Aftertaking brightfield and fluorescent images of the transfected sample all cellsare counted in brightfield and fluorescent images Transfection efficiency is computed by dividing the number of fluorescent cells by the total number of cells Note Assay parameters for assays that are included in the software are locked and can not be edited To customize this assay to suit your partic
25. Assay Button The yellow pencil next to Assay SETUP Assay Mame Assay 02 Trypan Blue Viability Assay is locked from editing Save as New Assay Type Special Cells Description Cell concentration and viability using trypan blue Imaging Mode Brightfield BR Only iv Trypan Blue Viability Fi Image Cell Type Celline Assay 02 el Description new cell type Edit Check Trypan Blue Viability 2009 Nexcelom Bioscience LLC Vision Manual 33 Tutorials Step 4 Click Preview Brightfield Image aag Live Cell Count eeh Trypan Blue Dead Tint F View Image Cell Type Imaging Mode Bnghtfield Only s S mage mage F2 image SAMPLE Sample ID Count In View i Manual Adjust LIL r Counted 293 Trypan Blue X Tee Dilution o Step 5 Input Dilution factor Dilution 2 if one part sample isadded to one part trypan blue This will ensure that counting results reflect cell concentration of original sample SAMPLE Sample ID 293 Trypan Blue Step 6 Adjust focus if necessary Step 7 Click Count 34 2009 Nexcelom Bioscience LLC Assay 02 Trypan Blue Viability Step 8 Review Counting Results and display data asneeded Counting Results Sample 293 Trypan Blue Dilution 2 0 Assay Assay 02 Trypan Blue Viability Description Cell concentration and viability using trypan blue Cell Cell Line Assay 02 Description new cell
26. Assay HUA Cell Line Assay HUA Cell Line GFP Cell Line_GFP Cell Line Medium Import Cell Line Medium Cell Line No decluster All gt gt Cell Line No decluster Cell Line Mo decluster Assay HU Cell Line No decluster Assay HU Cell H Cell H L Hand Pl L Hand Pl CHond_trypan CHond_trypan ES Cell FF ES Cell YFP Fl only Fl only w Drop down menu options New Cell Type Edit Highlighted Delete Highlighted Clear All Export to Create Library See Nexcelom Cell Library built in cell library from Nexcelom Browse locate user defined Cell library Cell Types in Cell Library list of Cell Types in a Cell Library Cell Types in Drop down Menu list of Cell Types in the drop down menu located in the Assay Type Import Highlighted import highlighted cell type from a Cell Library Import All import all cell types from the Cell Library Drop down menu option check to see details jw Drop down menu options New Cell Type E dit Highlighted Delete Highlighted Clear All Export to Create Library 100 2009 Nexcelom Bioscience LLC Software Operation Overview New Cell Type start a new cell type from default setting Edit Highlighted edit the highlighted celltype Delete Highlighted delete highlighted cell type Clear All clearall cell typesin the drop down list Export to Create Library generate a new Cell Type Library from the list of cell types Done close Cell Type Manager O 2009 Nexcelom Bioscience LLC
27. Before using Cellometer Vision for running samples use the training bead solution to ensure the instrument is setup and configured properly Step 1 Vortex Training Bead Solution at low speed for 10 seconds Remove a clean Disposable Counting Chamberand place on a clean Kimwipe Pipette all 20uL of solution into one sde of the chamber Capillary force automatically soreads the sample within the counting chamber Hold the loaded chamberin the white area taking care to stay away from the clearoptical window area Insert chamber into slot in the front of the instrument Step 2 Select Training Beads from the Assay type drop down menu RS ASSAY Set HES Gell Type Training Beads Imaging Mode Brightfield and Fluorescence Step 3 Click Preview Bnghtfield Image You should see an image similarto the one below 2009 Nexcelom Bioscience LLC Vision Manual 21 Getting Started Step 4 Slowly tum the black focus wheel until optimal cell counting focus is achieved NOTE Live cells and training beads wil have a bright centerand a cleardefined edge Step 5 Click Preview F1 Image You should see an image like the one below Step 6 Click Count or Soeed Count to begin the counting process o 22 2009 Nexcelom Bioscience LLC Using Vision forthe First Time Step 7 The counting results box will display upon conclusion of the counting process You should see ce
28. Cellometer Vision User Manual 2009 Nexcelom Bioscience LLC Technical Support Nexcelom Technical Support is available to assist with any technical issues or questions you might have during the installation or operation of the Cellometer Vision Email support nexcelom com Phone 1 978 327 5340 Fax 1 978 327 5341 Technical Support is available Monday Friday 8 00 AM to 5 00 PM Eastern US Time Warranty Information Nexcelom warrants that Nexcelom instrumentation products shall for a period of 12 twelve months from the date of purchase be free of any defect in material and workmanship The sole obligation of this warranty shall be to either repair or replace at our expense the product at manufacturers option The original sales receipt must be supplied for warranty repair Products which have been subjected to abuse misuse vandalism accident alteration neglect unauthorized repair or improper installation will not be covered by warranty Any Product being returned is to be properly disinfected and packaged in original packing if possible Damage sustained in shipping due to improper packing will not be covered by warranty A valid Return Material Authorization Number RMA is required for all warranty repairs For RMA instructions please contact our customer service department at 978 327 5340 or email support nexcelom com License Agreement This agreement states the terms and conditions upon which Nexcelom B
29. Counted check to see counted cells outlined When both fluorescence channels are used check the Combined box to display an overlay of F1 and F2 captured images o Oo s Go OQ Co A o 9 Uu a g i EI o5 o o om 3 CH Ri Mm oO A A Cell Image before counting B Counted cell image Green outline indicate counted cells 32 2009 Nexcelom Bioscience LLC Assay 02 Trypan Blue Viability Assay 02 Trypan Blue Viability Trypan blue is routinely used to determine cell viability Trypan blue penetrates and stains dead cells and leaveslive cells unstained After taking bnghtfield images of the stained sample live and dead cells are counted to determine total live and dead cell concentrations as wellascompute percent viability Note Assay parameters for assays that are included in the software are locked and can not be edited To customize this assay to suit your particularapplication please see Modify Default Assay Parameters for instructions on how to modify and save aSa new assay Step 1 Mix cell sample with trypan blue and incubate using the same method as when manually counting Step 2 Load 20uL of cell sample in one side of the cell counting chamberand insert into instrument Step 3 Select Assay 02 Trypan Blue Viability from Assay SETUP drop down menu SETUP Assay Assay 02 Try Cell Type Cell Line Assay 02 Z Imaging Mode Brghtfield Only Image mode Brightfield Only To review Assay Type details click Edit
30. SC C WEE in hole Blood Assay S07 PIC wth AO Asie EOS PI Mate Asa Basay 206 Caen AH Marga Assay 10 PL Sperm Assay za AOP We Age 11 AGPt Splenocyte G ing 12 ACER Seder te Air 12 ACER Spenocybe Bead FAI Beads PU Bead P 8 DEL A Beads FL1 amp F 2 Beads 52 Beads PLZ FF Drop down menu epson Delete Highlighted Nexcelom Assay Library default library from Nexcelom Browse select location for userto save Assay Library Assay in Assay Library list of Assay Types in an Assay Library Assays in Drop down Menu List of Assays in the Vision software Setup Assay Drop down list Import Highlighted Import highlighted assay type in a selected assay library to the Assay drop down menu in Setup Import All import the complete list of Assa ys from the Assay Library to the drop down menu Drop down menu options de selectto hide delete and export functions Delete Highlighted Clear All Export to Create Library v Drop down menu options Delete Highlighted delete highlighted assay type Clear All clearall assay typesin the drop down menu Export to Create Library export all the assay types in the drop down menu to a new Assay Library Done close Assay Manager 96 2009 Nexcelom Bioscience LLC Software Operation Overview Assay Type gt Edit Assay Type Assay Type Assay Name Assay 03 GFP Transfection Rate Assay is locked from editing Save as New Assay Type Spedal Cells Description Cell
31. Vision Manual 101 Operation Reference Options Menu Options Help Counting Optians Exposure Adjustment b Instrument Options Counting Options Counting Options Count All iw Speed Count Stop after Lells i Stop after 1 Images Intelligent File Load w Show Trypan Blue Dead Cell Count Flat Field Fluorescent images e Count All checkto count all four pre defined locationsinside each counting chamber Speed Count check for count without finishing all four pre defined locations Stop after of Cells check to stop counting after of user defined cells are counted and the next frame hasfinished counting Stop after of Images stop counting after finishing user defined images that are less than 4 images Show Trypan Blue Dead Cell Count check to display dead cell counts on the main software panel Use Background file with Fluorescence images allow fluorescence background image to be used fordata analysis OK close Counting Option and save changes Cancel close Counting Option menu without saving changes Options gt Take Background Image Take brightfiled background image of the Vision system without cell counting chamber For Vision Trio instruments Background Images are needed for both fluorescence channels Options Take Huorescence Background This function currently not available Options Exposure Adjustment Show Exposure Adjustment display Exposure time Save Exposure Time as Defaul
32. all available data display options 2009 Nexc elom Bioscience LLC Vision Manual 67 Tutorials Assay Type Assay Name Assay 08 PI Viability Jurkat Assay is locked from editing Save as New Assay Type Special Cells Description Call line viability using PI to stain dead cells Imaging Mode Brightfield BR amp Fluorescence F1 Trypan Blue Viability M Use BR Cell for F1 Count Fl Image Cell Type Jurkat PI Assay 08 Description Fluorophore Pr Ex 525nm 202 Fluorescent Exp 5000 0 msec Filter Set Protocol Notes Browse Hide Report Format SHOW M Show Cell Count show Trypan Blue Dead Celcom Iw Show Cell Mean Diameter Show Trypan Blue Dead Cell Mean Dian Iw Show Dilution MW Show Concentration Iw For Fluorescent Positive Cells Use BrightField Cell Size for Histogram Separate Fluorescent Positve Megative Cells for Cell Size Histogram Show Percent Fi BR Iw Show Sample Adjustment MW Show Cell Size Distribution M Show Cell Intensity Distribution M Show Cell Size Intensity Distribution MW Show Data File Buttons Step 4 Click Preview Bnghtfield Image Step 5 Adjust focus if necessary Step 6 Click Count 68 2009 Nexcelom Bioscience LLC Assay 08 Cell Viability Using Propidium lodide Staining Step 7 Review Counting Results and display data asneeded Counting Results Sample Jurkat PI Viability Dilution 2 0 Assay Assay 08_PI Viability Jurkat D
33. assay from the drop down list or c zat Assay Type edit an existing assay File ESA Options Help 2 InputSample ID and Dilution Factor Sample ID HCT116 GFP 3 Prepare sample load disposable counting chamber and insert into instrument 4 Click Preview Bnghtfield Image and ensure image is in focus 5 Adjust Focus Slowly tum the black focus wheel until optimal cell counting focus is achieved Live cells and training beadswillhave a bnght centerand a clear defined edge 6 Click Preview F1 Image and adjust exposure time if necessary A Preview F1 Image If using Cellometer Vision Trio in dual fluorescence mode click Preview F2 Image E posure ms ml n and adjust exposure time if necessary 1000 0 meec 7 Click Count to begin counting process 2009 Nexcelom Bioscience LLC Vision Manual 9 Introduction 10 Review Counting Results and generate desired reports Counting results will automatically display on screen Select desired reporty gra phs to display export pnnt data orclick Done to clear window and review on screen images Navigate and review images on screen a Selecta section of the image to view b toggle between the bnghtfield BR and fluorescence F1 or F2 images or c manually adjust counted cells If desired click a Re display Counting Results D Launch Sample Adjustment Calculator or c Display Size Distribution Counted Count I
34. ation and Viability Using AO Pl 79 Assay 12 Splenoc yte Concentration and Viability Using AO EB 84 Modify Default Assay Parameters wine 90 Part IV Operation Reference 94 Software Operation Overview ce escena TT nuu nn Une 95 SIE NEU rm 95 NCC NICE LL LLL III LLL ETT 96 OD UO AS se esed ve nat nie rate eee mt ennemi ue 102 a UE 104 2009 Nexcelom Bioscience LLC 3 Vision Manual Part V Technical Information 106 SpecificatioNS s mmsssssmssrrrrnennnnnnnnnnnnnnnnnnnnnnnnennnnnnnnnnnnnnn nn 107 Getting SUPpOrt nnn nee 108 Warranty Information 109 4 2009 Nexcelom Bioscience LLC Introduction Introduction 6 2009 Nexcelom Bioscience LLC What is Cellometer Vision What is Cellometer Vision Cellometer Vision isa compact automated cell analysis system that can count cells measure cell sizes and detect fluorescence properties of cells The basic principle of the Cellometer automatic cell counter is imaging cytometry Cells are loaded into the Disposable Counting Chamberand automatically spread into a thin layer by capillary action Cellometer Vision then captures images of cells in the counting chamber analyzesthe number of cells sizes and fluorescence intensity of each cell then convertsthis data into concentration size and fluorescence histograms and scatter plots The Cellometer Visio
35. ault Assay Parameters for instructions on how to modify and save asa new assay Step 1 Dilute blood in PBS 1 10 Step 2 Mix cell samples with AO and incubate using the same method as when manually counting blood concentration 1 20 AO concentration 2 10 mg mL Step 3 Load 20uL AO stained cell sample in one side of the cell counting chamber and insert into instrument Step 4 Select Assay 06 WBC in Whole Blood from the Assay SETUP drop down menu Imaging Mode Fluorescence Only Imaging mode Fluorescence Only Click on the yellow pencil on the right side of Assay selection to view Assay Type Assay Name Assay 06 WBCin Whale Blood Assay is lacked from editing Save as New Assay Type Special Cells Description WBC stained with AQ in human whole blood without lysing RI Imaging Mode Fluorescence E 1 Only lw Take BR with FL images Use BR Cell for Fi Count r F1Image Cel Type e E WBC AO Assay 06 MM Description Fluorophore Fluorescent Exp 2000 0 mser Filter Set Take BR with FL Image check to take bnghtfield image of the same cell sample 2009 Nexc elom Bioscience LLC Vision Manual 59 Tutorials Use BR cell for F1 Count uncheckto NOTuse brightfield counting method Step 5 Click Preview Bnghtfield Image Step 6 Adjust focus if necessary Step 7 Click Preview F1 Image Step 8 Click Count Step 9 Review Counting Results and display data asneeded Counting Results Sam
36. d concentration or total cell number View Size Distnbution click to display size distnbution after the initial pop up box hasbeen cleared Recount click to recount the same acquired image after Assay or Cell parameters have been modified Green Circle Indicates a counted cell In brightfield mode indicates a live cell when a running Trypan Blue assays In fluorescent mode indicates a fluorescence positive cell Red Circle In brightfield mode indicatesa dead cell Trypan Blue Positive when running Trypan Blue assays In fluorescent mode indicates a fluorescence negative cell 2009 Nexc elom Bioscience LLC Vision Manual 13 Getting Started Getting Started 16 2009 Nexcelom Bioscience LLC Cellometer Vision System Components Cellometer Vision System Components USB 2 0 Cable Laptop Controller Cellometer Vision Power Supply The Cellometer Vision comes with the following components Cellometer Vision Instrument Laptop Controller and associated accessories and documentation USB 2 0 Cable Cellometer Vision Power Supply Cellometer Vision Software CD User sManual Consumable Starter Pack Disposable Counting Chamber Slides and Training Bead Solution O 2009 Nexcelom Bioscience LLC Vision Manual 17 Getting Started FRONT BACK VIEW Cooling Vent DO NOT BLOCK Disposable Focus Chamber Slot Adjustment Power switch USB Input Power adapter input
37. d repair or improper installation will not be covered by warranty Any Product being retumed isto be properly disinfected and packaged in original packing if possible Damage sustained in shipping due to improper packing will not be covered by warranty A valid Retum Material Authorization Number RMA is required forall warranty repairs For RMA instructions please contact our customer service department at 978 327 5340 or email support nexcelom com License Agreement This agreement states the terms and conditions upon which Nexcelom Bioscience LLC Nexcelom offers to license to you the software together with all related documentations The Software is licensed to you for use only in conjunction with Nexcelom s family of products Limitation of Liability Hardware and Software Cellometer branded automatic cell counting instruments software and consumablesare intended for research use only In no event shall Nexcelom be liable forany damages whatsoever including without limitation incidental direct indirect special or consequential damages damages for loss of business profits business interruption loss of business information arising out of the use or inability to use this Software Consumables orrelated Hardware 2009 Nexcelom Bioscience LLC Vision Manual 109 Nexcelom Bioscience LLC 360 Merrimack Street Building 9 Lawrence MA 01843 USA Phone 1 978 327 5340 Fax 1 978 327 5341 Email support nexce
38. dilution factor Previews bnghtfield image before counting Previews the fluorescent image F1 Adjusts exposure time for F1 image in milliseconds Previews the fluorescent image F2 Adjusts exposure time for F2 image in milliseconds Initiates counting procedure Recounts currently loaded image Openscounting results box Launches Sample Adjustment Calculator Openssize distnbution histograms Shows number of live cells in current view Shows number of Trypan Blue stained dead cells in current view Shows number of fluorescent positive cells in Fl image 2009 Nexc elom Bioscience LLC Vision Manual 11 Introduction 22 TintF Check to display fluorescent cells in false color 24 Captured Image Quadrant Buttons Toggles view between quadrant 1 2 3 and 4 of each captured image 26 Combine Displays an overlay of Fl and F2 images 28 Live Dead Countin View Displa ystotal number of live ordead counted cells in view 30 Enter Updatescounting results after entering Manual Adjust values 12 2009 Nexcelom Bioscience LLC Terms and Icons Used Terms and Icons Used Assay A set of configuration parameters that include the instrument settings i e fluorescence modes exposure times counting method and associated cell types Cell Type A setof configuration parameters specific to a cell used in an assay such ascell size Parameters fluorescence properties and decluster properties Decluster A software function used to dis
39. edited from the main screen by clicking on the pencil icon ssay Type Assay Name Assay 04 RFP_Transfection Rate Assay is locked from editing Save as New Assay Type Special Cells Description Cell line transfection rate measurement using RFP Imaging Mode Brightfield BR amp Fluorescence F1 Trypan Blue Viability v Use BR Cell for F1 Count Fi Image Cell Type Cell Line_Assay 04 id Description Edit Fluorophore RFP Ex_620 nm 202 e Fluorescent Exp 2000 0 msec Filter Set Protocol Notes Browse Show Report Format Show me 2009 Nexcelom Bioscience LLC Vision Manual 45 Tutorials Step 4 Click Preview brightield Image IU Cellometer Vision Trio 5 File Assay Type Options Help Cell Count F1 Count Tint F SETUP F View Image Assay N i TG rZ Cell Type Imaging Mode 1 9 9 H do BR Image Brightfield and Fluorescence F L F1 Image F2 image SAMPLE Sample ID P SF9 RFP Dilution Step 5 Adjust Focus if necessary Step 6 Click Preview F1 Image Fluorescence I Cellometer Vision Trio 5 File Assay Type Options Help Vision El EJ mA Cell Count F1 Count Tint F SETUP View Image Assay Cell Type Cell Line_Assay 04 rZ Imaging Mode em Brightfield and Fluorescence Sg F1 Image F2 image SAMPLE Sample ID SFORFP Counted Dilution Count In View Manual Adjust 2000 0 meee Step
40. ers for instructions on how to modify and save asa new assay Step 1 Prepare cells suspended in growth media or PBS Step 2 Load 20uL of cell sample in one side of the cell counting chamberand insert into instrument Step 3 Select Assay 01 Concentration from Assay SETUP drop down menu Cell Type Cell Line Assay 01 Imaging Mode Brightfield Only Step 4 Click Preview Brightfield image IN Cellometer Vision Trio 5 File Assay Type Options Help Tint F View Image Cell Type a Cell Line _Assay 01 Imaging Mode Brightfield Only Count In View Manual Adjust Step 5 Adjust focus if necessary Step 6 Click Count 28 2009 Nexcelom Bioscience LLC Step 7 Review Counting Results and display data asneeded Counting Results Sample HCT116 Total Count Dilution 1 0 Assay Assay 01 Concentration Description Total cell concentration without trypan blue Cell Cell Line Assay 01 Description Bright Field Count a 1048 d Show Size Distribution Mean Size b 12 0 Sample Adjustment Concentration 0 47 x10 E f Set Data File Save to Data File View Data File d Count total number of cells counted b Mean Size mean diameter of the counted cells c Concentration cell concentration mL Assay 01 Total Cell Concentration Measurement d Show Size Distribution displays cell size histogram generated using brightfield image e Sample Adjustment launch sample adj
41. es Open Bright Field Image A Look in E Assay_ Images do t Assay 01 Total Cell Concentration BrightField Only Assay 02 Trypan Blue Viability BrightField Only 3 Assay 03 Transfection Rate Grp BrightField amp Green Fluorescence Assay 04 Transfection Rate RFP BrightField amp Red Fluorescence Assay 05 Transfection Rate YFP BrightField amp Green Fluorescence Assay 06 WEBC in Whole Blood BrightField amp Green Fluorescence Assay 07 PBMC BrightField amp Green Fluorescence Assay 08 PI Viability Jurkat BrightField amp Red Fluorescence D Assay 09 Calcein AM Macrophage BrightField amp Green Fluorescence Assay 10 PI Sperm Red Fluorescence Assay 11 AOPI LiveDead Assay 12 AOEB LiveDead Bitmap file Vision BR A bmp Note Assay parameters for assaysthat are included in the software are locked and can not be edited To customize any assay to suit your particular application please see Modify Default Assay Parameters for instructions on how to modify and save asa new Assay 2009 Nexc elom Bioscience LLC Vision Manual 27 Tutorials Assay 01 Total Cell Concentration Measurement After taking brightfield images of a cell sample all cells are counted to determine total cell concentration Note Assay parameters for assays that are included in the software are locked and can not be edited To customize this assay to suit your particularapplication please see Modify Default Assay Paramet
42. escription Cell line viability using PI to stain dead cells Cell Jurkat PI Assay 08 Descriptian Bright Field Fluorescence Count 2028 is4 Show Size Distribution Mean 5ize 12 9 13 7 Intensity Distribution BR Size Size vs Intensity Concentration 167 x10 B f 134 x10 e SAREN E Sample Adjustment BR F1 Count g On g Set Data File BR Total Count Save to Data File View Data File ISS a Count Bright Field total number of cells counted in the brightfield image b Count Fluorescence number of fluorescence positive cells counted c Mean Size Bright Field mean diameter of cells measured in brightfield d Mean Size Fluorescence mean diameter of fluorescence positive cells e Concentration Bright Field total cell concentration mL of original sample f Concentration Fluorescence fluorescence cell concentration mL g BR F1 Count BR Total Count via bility of cell sample h Export storesall raw data assay parameters and graphsin an Excel file i Print send cument counting resultsto printer j Done closescounting results window Note After clicking Done the counting results box can be redisplayed atany time by clicking on the View Report of Counting Results button O 2009 Nexcelom Bioscience LLC Vision Manual 69 Tutorials Step 8 Review Imageson screen View Image Zoom In F1 Image F2 Image Combined Counted A one of the fourcaptured images 1 2 3
43. ew Data File save Images Save Counted Images Exit File gt Load Image for Display Load saved cell images for display When Assay Type includes both bnghtfield and fluorescence multiple images are required File gt Load Image and Count Load saved cell imagesforcounting When Assay Type includes both brightfield and fluorescence multiple images are required Saved cell images are counted using cument Assay Type parameters File gt New Data File Start a new text file for Cell Count Table File gt Select Data File Select a different text file for saving Cell Count Table File gt View Data File Display currently selected Cell Count Table File gt Save Images Save cell images File gt Save Counted Images Save cell images with outlines indicating counted objects File gt Save Combined Images Save the combined view showing F1 and F2 fluorescent objects in a single merged image File gt Exit Quit Cellometer Vision software 2009 Nexc elom Bioscience LLC Vision Manual 95 Operation Reference Assay Type Menu Edit Assay Type Delete Assay Type Assay Type gt Import Export Assay The Assay Type Library isa list of defined assay types Assay Type Manager is used to import from the existing assay library orto export a user defined list of assay types into a new Assay Library Assay Ty pe Munadger Assay 201 Concentrabon Assay 807 Trypan Bloe late Ass ejt E Transfecton Hate Assay
44. g chamberand insert into instrument Step 3 Select Assay 12 AOEB Splenocyte from the Assay SETUP drop down menu Imaging Mode Fluorescent 1 Fluorescent 2 Cell Type Splenocyte Assay 12 is displayed Imaging Mode Fluorescent 1 Fluorescent 2 The fluorescent 1 is used to measure live cells while the fluorescence 2 is used to measure dead c ells Both Cell Type and Imaging Mode are defined in the Assay parameters and can be shown from the main screen by clicking on the pencil icon 84 2009 Nexcelom Bioscience LLC Assay 12 Splenocyte Concentration and Viability Using AO EB Assay Name Assay 12 AOEB Splenocyte Assay is locked from editing Save as New Assay Type Special Cells Description Solenocyte stained with AOEB mixture to measure live amp dez 5 I Imaging Mode Fluorescence 1 F1 8 Fluorescence 2 F2 el Take BR with FL images Two Chamber Assay Use BR Cell for F1 Count r F1 Image 1 rFZImage Cell Type Splenocyte_Assay 12 Cell Type Splenocyte_Assay 12 Description Edit Description Edit Fluorophore AO 101 Fluorophore EB 702 Fluorescent Exp 1000 0 mser Filter Set Fluorescent Exp 2000 0 msec Filter Set Protocol Notes Browse Show Report Format Show Step 4 Input Sample ID and dilution factor SAMPLE Sample ID Splenocyte AOEB 2 Dilution Step 5 Input 2 for Dilution 2009 Nexcelom Bioscience LLC Vision Manual
45. gram Files Nexcelom Cellometer Vision intensity BRFL tat Cell Size Cell Intensity aa 3 Cell Size vs FL Intensity 12 34 12 61 10 92 15 23 15 34 7 28 9 46 10 61 12 21 12 87 11 93 13 25 11 93 12 61 9 46 10 92 ee 8 j Cell Size Microns 12 87 EC FSC a J Open Size File Open Intensity File Copy to Clipboard OO JO C CO P2 40 2009 Nexcelom Bioscience LLC k Sample Adjustment Calculator Sample Adjustment Measured Concentration cells ml 3 25e 005 ooo Total Cell Number in Sample 4 63e 006 Original Sample Yalume ml Iw Target Concentration cellz ml 1 00e 006 Target Number of Cells FOCDOL Assay 03 GFP Transfection Rate Sample Adjustment Measured Concentration cells ml 3 25e 005 59 Total Cell Number in S ample 4 63e 006 Original Sample Volume ml Target Concentration cells ml M Target Number of Cells 20000 ee E pe eee Apply nange PEDES aM et e Apply Lange Sample Adjustment Take 27 02 ul of sample Good for 185 aliquots Print with report Sample Adjustment Spin down and add diluent amount 463 ml Print with report Calculator 2 input target number of needed Amount of sample to add and the number of aliquots will be calculated Calculator 1 input target cell concentration and calculate adjustment n View Data table Cell Counting Table list saves counting results from multiple
46. gram generated using brightfield image i Intensity Distribution displays fluorescence cell intensity histogram j Size vs Intensity displays fluorescence intensity vs cell size scatterplot k Sample Adjustment calculates adjustment for target concentration ortarget number of cells L Set Data File creates a data file to store cell counting table m Save to Data File records counting results in the cell counting table n View Data File displa ys currently selected cell counting table o Export stores all raw data assay parameters and graphs in an Excel file p Print sendsthe cument counting resultsto a printer q Done closescounting results window Note After clicking Done the Counting Results box can be redisplayed at any time by clicking on the View Report of Counting Results button O 2009 Nexcelom Bioscience LLC Vision Manual 39 Tutorials h Cell size distribution for brightfield image Cell TETUR Analysis TTT TTT TTT ORES GRRE OTI Don Diaeta File NOTE this can be instantly displayed at any time by clicking on the _ icon i Fluorescence intensity versus number of cells Cell lilsmedby Analysis b emin File Hana Pg FlesiAHesirnlom Celiometer Viren k rerig BREL b Call Intensity Histogram Total Call Count Ch oC zz DDR ode LO Bo di Cell Size intensity Analysis Diameter File Name C Program Files Nexcelom Cellometer Vision diameters tat Intensity File Name C Pro
47. ioscience LLC Nexcelom offers to license to you the software together with all related documentation The Software is licensed to you for use only in conjunction with Nexcelom s family of products Limitation of Liability Hardware and Software Cellometer branded automatic cell counting instruments software and consumables are intended for research use only In no event shall Nexcelom be liable for any damages whatsoever including without limitation incidental direct indirect special or consequential damages damages for loss of business profits business interruption loss of business information arising out of the use or inability to use this Software Consumables or related Hardware All rights reserved No parts of this work may be reproduced in any form or by any means graphic electronic or mechanical including photocopying recording taping or information storage and retrieval systems without the written permission ol Nexcelom Bioscience LLC Other products that are referred to in this document may be either trademarks and or registered trademarks of the respective owners Nexcelom makes no claim to these trademarks While every precaution has been taken in the preparation of this document Nexcelom assumes no responsibility for errors or omissions or for damages resulting from the use of information contained in this document or from the use of programs and source code that may accompany it In no event shall the publi
48. ize C 13 1 d 13 3 Intensity Distribution BR Size j Size vs Intensity Concentration s 42 x10 f 3 31 x10 7 EL oe 795 Set Data File BR Total Count m Save to Data File n View Data File Count brightfield total number of cells counted in the brightfield image Count Fluorescence number of fluorescence positive cells counted Mean Size brightfield mean diameter of cells measured in brightfield Mean Size Fluorence mean diameter of fluorescence postive cells Concentration Bright Field total cell concentration mL Concentration Fluorescence fluorescence cell concentration mL F1 Count BR Total Count transfection rate gt 070000020 Show Size Distribution displays cell size histogram generated using brightfield image i Intensity Distribution displays fluorescence cell intensity histogram j Size vs Intensity displays fluorescence intensity vs cell size scatterplot k Sample Adjustment calculates adjustment fortarget concentration ortarget number of cells L Set Data File creates a data file to store cell counting table m Save to Data File records counting results in the cell counting table n View Data File displa ys cumently selected cell counting table o Export stores all raw data assay parameters and graphs in an Excel file p Print sendsthe cument counting resultsto a printer q Done closescounting results window Note After clicking Done the Counting Result
49. ll counts in both the brightfield and fluorescence boxes The results should be approximately what is pictured below If the these figures are grossly out of range please contact Nexcelom support at 1 978 327 5340 or supportinexc elom c om or your authorized distributor Counting Results Sample Training Beads Dilution 1 0 Assay Training Beads LIESCrID Show Size Distribution Intensity Distribution BR Size Size vs Intensity ee 1 23 x10 eats eee Concentration 3 30 x10 5 F1 Count NN E 37 5 Set Data File BR Total Count Save to Data File Export View Data File mm Initial setup and configuration are now complete You are ready to start using Cellometer Vision Section Ill contains tutorials of common applications to help familiarize yourself with the instrument and its functions O 2009 Nexc elom Bioscience LLC Vision Manual 23 Tutorials Tutorials 26 2009 Nexcelom Bioscience LLC Tutonal Overview Tutonal Overview The following tutorials are intended asa guide to performing vanous cell based assays using the Vision General sample preparation hints are included for each tutorial as well as instrument and software operation instructions Each of the assays can also be perfomed using the sample images included in the software asa demonstration of instrument and software operation Sample images foreach assay can be found at C Program Files Nexcelom Vision Assay Imag
50. ll concentration measured by Cellometer Original Sample Volume total volume of cell sample Total Cell Number in Sample total number of cells in the onginal sample Sample Adjustment instructions for sample adjustment to obtain desired results Sample Adjustment sample Adjustment Measured Concentration cells ml 1 89e 006 Measured Concentration cells ml 1 89e 006 Original Sample Volume ml 10 0 Original S ample Volume rm 10 0 Total Cell Number in Sample 1 89e 007 Total Cell Number in Sample 1 89e 007 W Target Concentration cells ml 1 00e 00 Sample Adjustment Add diluent amount 6 94 ml Target Humber of Cells Target Concentration cells ml Iw Target Number of Cells 100000 Sample Adjustment Take p2 A0 ul af sample Good for 183 aliquots M Print with report Ok M Print with report Calculator 1 adjust to obtain target cell Calculator 2 adjust for target number of concentration cells Target Concentration desired cell Target Number of Cells desired number of concentration mL cells Apply Change to calculate adjustment 2009 Nexcelom Bioscience LLC Vision Manual 31 Tutorials Step 8 Review Imageson screen Zoom in 61 Image F1 Image F2 Image Combined Counted A one of the fourcaptured images 1 2 3 or 4 one quadrant of a selected image FL fluorescence 1 image view F2 fluorescence 2 image view Combined check to display an ovenay of Fl and Fl images
51. ll sample with AO and incubate using the same method as when manually counting AO concentration 2 10 mg mL Step 2 Load 20uL in one side of the cell counting chamber and insert into instrument Step 3 Select Assay 07 PBMC with AO from the Assay SETUP drop down menu Cell Type PBMC AO A Imaging Mode Fluorescence Only Image mode Fluorescence Only Click on the yellow pencil on the nght side of Assay selection to view Assay Type Assay Name Assay 07 PBMC with AO Assay is locked from editing Save as New Assay Type Special Cells Description PBMC stained with AO without lysing RBC Imaging Mode Fluorescence F1 Only fw Take BR with FL images Use BR Cell for F1 Count r F1Image Cell Type PBMC AO Assay 07 Description Edit Fluorophore AO Ex 475 nm 101 Fluorescent Exp 2000 0 mser Filter Set Take BR with FL Image check to take bnghtfield image of the same cell sample Use BR cell for F1 Count uncheckto NOTuse brightfield counting method O 2009 Nexcelom Bioscience LLC Vision Manual 63 Tutorials Step 4 Click Preview Brightfield Image Step 5 Adjust focus if necessary Step 6 Click Preview F1 Image Step 7 Click Count Step 8 Review Counting Results and display data asneeded Counting Results Sample PBMC with RBC Dilution 1 0 Assay Assay 07 PBMC with AO Description PBMC stained with AO without lysing RBC Cell PBMC AO Assay 207 Description F
52. lom com www nexcelom com All content copyright 2008 Nexcelom Bioscience LLC Rev 020609
53. luorescence Count a C Concentration 1 46 x10 Sample Adjustment Set Data File d cave to Data File Export View Data File a Count number PBMC counted b Concentration PBMC concentration mL c Sample Adjustment calculates adjustment fortarget concentration or target number of cells d Save to Data File record counting resultsin a table e Export stores all raw data assay parameters and graphsin an Excel file f Print send current counting results to printer g Done closes counting results window Note After clicking Done the counting results box can be redisplayed at any time by clicking on the View Report of Counting Results button 64 2009 Nexcelom Bioscience LLC Assay 07 PBMC Concentration Using Acridine Orange Staining Step 9 Review Images on screen View Image Zoom In ET Image Fi Image F2 Image v Combined EJ Counted A one of the fourcaptured images 1 2 3 or4 one quadrant of a selected image FL fluorescence 1 image view F2 fluorescence 2 image view Combined check to display an overay of Fl and Fl images Counted check to see counted cells outlined When both fluorescence channels are used check the Combined box to display an overlay of F1 and F2 captured images A Brightfield image of PBMCs with contaminating red blood cells 2009 Nexcelom Bioscience LLC Vision Manual 65 Tutorials B Fluorescence image of AO stained C
54. n Fluorescence Count a 488 Intensity Distribution Sample Adjustment Concentration b 1 37 x10 5 Set Data File Save to Data File View Data File a Count Fluorescence number of fluorescence positive cells counted b Concentration Fluorescence fluorescence cell concentration mL c Export sores all raw data assay parameters and graphsin an Excel file d Print send cument counting resultsto printer e Done closescounting results window Note After clicking Done the counting results box can be redisplayed atany time by clicking on the View Report of Counting Results button 2009 Nexc elom Bioscience LLC Vision Manual 77 Tutorials Step 8 Review Imageson screen Zoom In El Image F1 Image F2 Image Combined Counted A one of the fourcaptured images 1 2 3 or 4 one quadrant of a selected image FL fluorescence 1 image view F2 fluorescence 2 image view Combined check to display an ovenay of Fl and F1 images Counted check to see counted cells outlined When both fluorescence channels are used check the Combined box to display an overlay of F1 and F2 captured images A Cell image before counting B Cell image after counting Green outline indicates sperm cells counted 78 2009 Nexcelom Bioscience LLC Assay 11 Splenocyte Concentration and Viability Using AO PI Assay 11 Splenoc yt Concentration and Viability Using AO PI Acridine orange isa nuclear stain
55. n View Manual Adjust BR Image F1 Image 10 2009 Nexcelom Bioscience LLC Software Overview 15 16 File Assay Type Options Help SETUP Assay d EES Cell Type i Splenocyte_Assay 11 ff Imaging Mode Fluorescent 1 Fluorescent 2 SAMPLE Sample ID 6 new sample ND AO 7 HAT Bi UU Et 9 10 Exposure ms oo 10000 meee 1 12 Exposure ms mmj 2000 0 meet Item Name Select Assay Modify Assay Cell Type Display Modify Cell Type Imaging Mode Display Sample ID Input Dilution Factor Input Preview Bnghtfield Image Preview F1 Image F1 Exposure Adjustment Preview F2 Image F2 Exposure Adjustment Count or Speed Count Button Rec ount Button Display Counting Results Sample Adjustment Calculator Display Size Distirbution Live Cell Count Display Trypan Blue Dead Display e PS bb So om P WM o amp S 20 F1 Counted Software Overview 18 19 20 21 Live Cell Count Trypan Blue Dead Tint F 22 View Image 23 24 BR Image F1 Image 25 F2 Image Combined 26 Counted 27 Live Dead Count In View sis 28 Manual Adjust EIE 29 Ener Ka What it does Selects a saved assay from the Assay Library Modifies parameters of currently selected Assay Displays the selected celltype Modifies parameters of currently selected Cell Type Displays current imaging mode settings Allows user to input name ID of current sample Allows user to input
56. n system consists of 3 main components 1 Cellometer Vision instrument e P t 2 Cellometer Vision Software and Controller Laptop Included with instumentand pre loaded with software 3 Disposable Counting Chambers 5 e Each disposable counting chamberaccommodates 2 individual samples and can be loaded through either port e Simply pipette 20uL into one of the ports with any standard single channel pipette e Cellometer Vision comes with a starter set of 25 slides Slides can be ordered directly from Nexcelom or your authonzed Nexcelom dealer 2009 Nexcelom Bioscience LLC Vision Manual 7 Introduction Generating Data with Cellometer Vision Generating cell sample information is quick and easy Once the instrument is setup for your particular assay only 3 basic steps are needed to get results imal e Trein Be nm Crete 72e 1 ETA eege Ka F a ie PL Cari BA Tom Caen n Tee EE me l prepare sample and load 2 Insert counting chamber into 3 Use Software to acquire images counting chamber instrument analyze samples and view results 8 2009 Nexcelom Bioscience LLC Quick Operation Instructions Quick Operation Instructions Below are the basic steps that are followed to run any assay Sep Instructions 1 Setup orSelect an Assay M Cellometer Vision SETUP Assay a Import or Setup a new assay from the RETIA Lo Assay Type drop down menu b Selectan existing
57. ned Counted A one of the fourcaptured images 1 2 3 or 4 one quadrant of a selected image FL fluorescence 1 image view F2 fluorescence 2 image view Combined check to display an ovenay of Fl and F1 images Counted check to see counted cells outlined When both fluorescence channels are used check the Combined box to display an overlay of F1 and F2 captured images 2009 Nexcelom Bioscience LLC Assay 11 Splenocyte Concentration and Viability Using AO PI B F2 C Combined D Combined and Counted 2009 Nexcelom Bioscience LLC Vision Manual 83 Tutorials Assay 12 Splenoc yte Concentration and Viabilrty Using AO EB Acridine orange isa nuclearstain that emits in the green range and isused to stain live cells Ethidium bromide isa fluorescent stain that only penetrates dead cells and emits in the red range Aftertaking both green and red fluorescent images all fluorescent cells in each channel are counted and the concentration of live green fluorescent and dead red fluorescent cells as well as viability are determined Note Assay parameters for assays that are included in the software are locked and can not be edited To customize this assay to suit your particularapplication please see Modify Default Assay Parameters for instructions on how to modify and save asa new assay Step 1 Mix 10 ul splenocyte sample with 10 ul AO EB mixture Step 2 Load 20 ul in one cell countin
58. nsity Histogram Number of Cells Cell Intensity Histogram Total Cell Count pen Intensity File Multiple Save As Copy to Clipboard j Fluorescence intensity versus cell size Cell Size Intensity Analysis Diameter File Name C Documents and Settings All Users pplication Data Nexcelom_Vision5 diameters txt Intensity File Name C Documents and Settings All Users Application Data Nexcelom_Vision5 intensity BRFL txt Cell Size Cell Intensity Cell Nu Cell Size Cell Number Intensit CO CO 7 D CD FR CO M CO CO JO CD FR CO M Cell Size Microns Open Size File Open Intensity File Copy to Clipboard 48 2009 Nexcelom Bioscience LLC Assay 04 RFP Transfection Rate k Sample Adjustment Calculator Sample Adjustment sample Adjustment Measured Concentration cells ml 1 676 006 Measured Concentration cells ml 1 87e 006 Original 5 ample Volume ml 1 0 Original Sample Volume ral 1 0 Total Cell Humber in 5 ample 1 87e 006 Total Cell Number in Sample 1 57e 00 W Target Concentration cells ml 1 00e 006 Target Concentration cells ml Target Number of Cells iw Target Number of Cells 100000 E d Sample Adjustment Sample Adjustment Add diluent amount 0 87 mil Take 53 36 ul of sample Good for 18 aliquots i Print with report Iw Print with report Calculator 1 input target cell Calculator 2 input target number of concentration and calculate needed
59. ode E BR Image Brightfield and Fluorescence e F1 Image F image SAMPLE Sample ID HCT116 GFP Dilution Count In View Manual Adjust Exposure ms Ge 2000 0 meee s 1d Step 7 Click Count 38 2009 Nexcelom Bioscience LLC Assay 03 GFP Transfection Rate Step 8 Review Counting Results and display data asneeded Counting Results Sample HCT116 GFP Dilution 1 0 Assay Assay 03 GFP Transfection Rate Description Cell line transfection rate measurement using GFP Cell Cell Line Assay 03 Description Bright Field Fluorescence Count a 104 Show Size Distribution 048 b 512 Mean Sire Le 12 0 d 12 1 Intensity Distribution E BR Size Size vs Intensity Concentration Ea x10 5 f 4 63 x10 5 Sample Adjustment F1 Count Cj A rA M ANT aT Set Data File i BR Total Count i i i Save to Data File Export O n View Data File Print Count brightfield total number of cells counted in the bnghtfield image Count Fluorescence number of fluorescence positive cells counted Mean Size brightfield mean diameter of cells measured in brightfield Mean Size Fluorence mean diameter of fluorescence postive cells Concentration Bright Field total cell concentration mL Concentration Fluorescence fluorescence cell concentration mL g Fl Count BR Total Count transfection rate dd Be ae ds h Show Size Distribution displays cell size histo
60. ons Filter set 1 188 88 I c 66 i T 5 4g m 2g 8 406 456 Ba 556 688 Havelength nnl Blue LED Excitation 470nm FITC Ale xa Fluor 488 Excitation 495nm Emission 519nm Acridine Orange DNA Excitation 500nm Emission 526nm Filter Set 2 188 8B I c 66 E b T u AB m E 26 458 588 2258 688 658 88 Havelength na Green LED Excitation 525 Propidium lodide PI Excitation 536 Emission 617 O 2009 Nexcelom Bioscience LLC Vision Manual 107 Technical Information Getting Support Technical Support Nexcelom Technical Support is available to assist with any technical issues or questions you might have during the installation oroperation of the Cellometer Vision Email supportanexcelom com Phone 1 978 327 5340 Fax 1 978 327 5341 Technical Support is available Monday Friday 8 00 AM to 5 00 PM Eastem US Time 108 2009 Nexcelom Bioscience LLC Warranty Information Warranty Information Warranty Information Nexcelom warrants that Nexcelom instrumentation products shall fora period of 12 twelve months from the date of purchase be free of any defect in material and workmanship The sole obligation of this warranty shall be to eitherrepairorreplace at ourexpense the product at manufacturersoption The original sales receipt must be supplied for warranty repair Products which have been subjected to abuse misuse vandalism accident alteration neglect unauthorize
61. or 4 one quadrant of a selected image FL fluorescence 1 image view F2 fluorescence 2 image view Combined check to display an ovenay of Fl and F1 images Counted check to see counted cells outlined When both fluorescence channels are used check the Combined box to display an overlay of F1 and F2 captured images a 9 g a e 9 v o a o Vy 9 o o o 4 o o s be D e z o H a o d on a e a QO d 9 A Ho y Do Va e o e s x 8 5 i 4 o B Counted cell image Green outline A Brightfield cell image indicates counted cells 70 2009 Nexcelom Bioscience LLC Assay 08 Cell Viability Using Propidium lodide Staining C Pl positive fluorescence cell image D Counted fluorescence cell image Green outline indicates PI positive cells O 2009 Nexcelom Bioscience LLC Vision Manual 71 Tutorials Assay 7 09 Live Cell Concentration using Calcein AM Calcein AM is used to determine cell viability and other key functional activities of cells Calcein AM is non fluorescent but when taken up by live cells it is converted to calcein a compound that emits in the green fluorescent range Aftertaking green fluorescent images all fluorescent cells are counted and their concentration in the sample is determined Brightfield images can also be taken and counted However in the sample images forthis assay brightfield counting picked up debristhat was eliminated by imaging in the fluorescent channel
62. ple WBC in whole blood Dilution 1 0 Assay Assay 06 WBC in Whole Blood New Description WBC stained with AO in human whole blood without lysi Cell WBC AO Assay 06 Description Fluorescence Count 136 C Sample Adjustment Set Data File Save to Data File em Export View Data File f Print g Done a Count total number of white blood cells b Concentration concentration of white blood cells c Sample Adjustment calculates adjustment for target concentration ortarget number of cells d Save to Data File record counting resultsin a table e Export storesall raw data assay parameters and graphsin an Excel file f Print send current counting results to printer g Done closescounting results window Note After clicking Done the counting results box can be redisplayed at any time by clicking on the View Concentration 7 76 x10 3 Report of Counting Results button 60 2009 Nexcelom Bioscience LLC Assay 06 White Blood Cell Concentration in Whole Blood with Acndine Orange Staining Step 10 Review Images on screen View Image D Zoom In 61 Image F1 Image F2 Image iv Combined v Counted A one of the fourcaptured images 1 2 3 or 4 one quadrant of a selected image FL fluorescence 1 image view F2 fluorescence 2 image view Combined check to display an ovenay of Fl and F1 images Counted check to see counted cells outlined When both fluorescence channels
63. r 4 one quadrant of a selected image FL fluorescence 1 image view F2 fluorescence 2 image view Combined check to display an ovenay of Fl and F1 images Counted check to see counted cells outlined When both fluorescence channels are used check the Combined box to display an overlay of F1 and F2 captured images e La Ge UU SE du v ct A Brightfield image of cells B Brightfield after counting This cell sample containsa large amount of debris which makesthe brightfield counting method less accurate 74 2009 Nexcelom Bioscience LLC Assay 09 Live Cell Concentration using Calcein AM C Fluorescence image of Calcein positive D Fluorescence positive cells after counting cells 2009 Nexcelom Bioscience LLC Vision Manual 75 Tutorials Assay 10 Spem Cell Concentration Using Propidium lodide Staining In this assay propidium iodide staining is used to count all cells in a sperm sample PI isa fluorescent stain that emits in the red range and isused to mark all soem after permeabilization After taking red fluorescent images all fluorescent cells are counted and their concentration in the sample is determined This assay demonstratesthat the Cellometer software can be used to count non circular objects such as sperm Note Assay parameters for assays that are included in the software are locked and can not be edited To customize this assay to suit your particularapplication please see Modify Defaul
64. s New Assay Type Description Solenocyte stained with AOPI mixture to measure live amp dea Imaging Mode Fluorescence 1 FDA Fluorescence 2 F2 Take BR with FL images Two Chamber Assay Use BR Cell for Fi Count F1 Image p F2 Image Cell Type Splenocyte Assay 14 Cell Type Splenocyte Description Description Special Cells Edit Fluerophore AO Fluarophore PI 202 Fluorescent Exp i000 0 meer Filter Set Fluorescent Exp 000 0 msec Filter Set Protocol Notes Browse Show Report Format Show an v Step 4 Input Sample ID and dilution factor SAMPLE Sample ID Splenocytes AOPI Live amp Deac Dilution Step 5 Input 2 for Dilution Step 6 Review cell images Preview B1 Freview F1 Exposure m 1002 of Range 1000 0 msec Preview F2 Exposure mz ml aaa 20000 mzec Click BT Preview BI will display brightfield image of F1 channel Adjust Focus if necessary Stop Review Click Preview EU Fluorescence 100 of Range indicates adequate exposure time for Fl channel g Click Preview F2 Fluorescence hm OO CO O 2009 Nexcelom Bioscience LLC Assay 11 Splenocyte Concentration and Viability Using AO PI Cellometer Warning Fluorescent intensity is only 14 of the full range Consider increasing the exposure time by a min of 6 5x h If exposure time is inadequate increase exposure time by using the pencil
65. s box can be redisplayed at any time by clicking on the View Report of Counting Results button 54 2009 Nexcelom Bioscience LLC Assay 05 YFP Transfection Rate h Cell size distribution for brightfield image Cell Size Analysis Diameter File Name C Documents and Settings 4ll Users Application Data Nexcelom_Vision5 diameters F1 txt Cell Diameters Histogram i Diameter um 1 2 3 4 5 6 7 8 g Percent 95 Total Cell Count Mean Diameter Open DiameterFile Multiple Save s Copy to Clipboard NOTE this can be instantly displayed at any time by clicking on the Lil icon i Fluorescence intensity versus number of cells Cell Intensity Analysis Intensity File Name C Documents and Settings All Users Application Data Nexcelom_Vision5 intensity BRFL txt Cell Intensity Histogram Cell Intensity Histogram Total Cell Count LE Open Intensity File Multiple Save s Copy to Clipboard Cancel j Fluorescence intensity versus cell size Cell Size Intensity Analysis Diameter Fie Name C Documents and Setings AllUsers Applicaion Dat Nexcelom VisonSidiameterstt it Intensity File Name C Documents and Settings 4ll Users pplication Data Nexcelom_Vision5 intensity BRFL txt Cell Size Cell Intensity Cell Nu Cell Size L 1413 Cell Size vs F1 Intensity 2 15 34 F 3 11 74 4 14 26 5 11 55 6 11 64 7 10 60 8 10 99 g 12 00 10 11 74 11 14 11 12
66. samples M View Data Data File C Cellometer Vision Vision Data T able txt Cell Count Dilution Factor Result Type Result Mean Diameter 07 16 2008 03 00 52 283 Trypan Viability 5 02e 005 Trypan Via 73 4 Cancel 0716 2008 09 00 52 283 Trypan Viability 1 82e 005 TrypanDead 26 6 07 16 2008 09 02 05 HCT116_Total Count 9 25e 005 07 16 2008 09 04 23 HCT116 GFP 9 25e 005 ES 07 16 2008 09 04 23 HCT116 GFP 5 42e 005 58 6 To Excel Open Excel o Export all raw data assay type cell type etc into an excel file Excel Export Option e Export Count details to Excel M Save Images with file M Save Counted Images with file M Open Excel when Done C Save Counting results to file f ps heme eile Erang e ME Continue Cancel NOTE User can select which items get exported to Excel 2009 Nexcelom Bioscience LLC Vision Manual 41 Tutorials Step 9 Review Imageson screen View Image Zoom In F1 Image F2 Image Combined Counted A one of the fourcaptured images 1 2 3 or 4 one quadrant of a selected image FL fluorescence 1 image view F2 fluorescence 2 image view Combined check to display an ovenay of Fl and F1 images Counted check to see counted cells outlined When both fluorescence channels are used check the Combined box to display an overlay of F1 and F2 captured images d o O o o 9 O O o O 9 o o 915 9 O A A Brightfield cell image B
67. sher and the author be liable for any loss of profit or any other commercial damage caused or alleged to have been caused directly or indirectly by this document Table of Contents Parti Introduction 6 What is Cellome WT E 7 Quick Operation Insiuc Bons sees ened 9 Software Overview sscsssssssseeeeeeeuennnaeeeeeeueennuaagseseeeeuaaauageseeeeuaeauaaaaseeeeeaeaaaagsseesennaanaaaaas 11 Terms and Icons Used ee 13 Part Il Getting Started 16 Cellometer Vision System Components 17 Setting up Cellometer VISION 19 Initial Configuration Setting Background Image nmmnnnnnnnnnns 20 Using Vision for the First Time nnn 21 Part Ill Tutorials 26 TubnalOVervieun usssssxessusuuxzuuunSa EES KEE ENEE Eege defgeeeEe Gu RENE EUEUNEKE NEN RE EE 27 Assay 01 Total Cell Concentration Measurement 28 Assay 02 Trypan Blue Viability ene 33 Assay 03 GFP Transfection Rate eene 37 Assay 04 RFP Transfection Rate nennen nnn nnmnnn 45 Assay 05 YFP Transfection Rate nnn 52 Assay 06 White Blood Cell Concentration in Whole Blood with Acridine Orange Staining 59 Assay 07 PBMC Concentration Using Acridine Orange Staining 63 Assay 08 Cell Viability Using Propidium lodide Staining 67 Assay 09 Live Cell Concentration using Calcein AM 72 Assay 10 Sperm Cell Concentration Using Propidium lodide IU A U 76 Assay 11 Splenocyte Concentr
68. t save an Exposure time that hasbeen changed as default When the Vision software is closed and restarted the saved exposure time will be used Options gt Instrument 102 2009 Nexcelom Bioscience LLC Software Operation Overview Instrument Options TN Note These are factory settings and are only editable when changing hardware configuration Please contact Nexcelom foradditional information Vision Duo Vision Trio Indicates which instrument the software is configured for Factory Setting Left L Filter Set Right R Filter Set Displays Filter Sets installed in the machine Vision Trio Only O 2009 Nexc elom Bioscience LLC Vision Manual 103 Operation Reference Help Menu Vision Help About Nexcelom Vision V1 1 0 Help Vision Help User manual and Tutorials Help About Nexc elom Vision V1 1 0 Displa ys version number of Cellometer Vision software currently installed Cellometer Vision Auto Counter Cellometer Vision Auto Counter Version 1 1 0 Copyright C 2005 2009 Nexcelom Bioscience LLC 104 2009 Nexcelom Bioscience LLC Technical Information Technical Information 106 2009 Nexcelom Bioscience LLC Specifications Specifications Spec ific ations Instrument Specifications Weight 25 lbs 11kg Dimensions 6 x85 x 14 15cmx22cmx36c m Wall Adaptor 100 240 AC 50 60 Hz 0 8A Powerto instrument 12 V DC 2 25 A Max Huorescence Specificati
69. t 1 indicates live cell concentration mL d Concentration Fluorescent 2 indicatesdead cell concentration mL e F1 Count F1 F2Count indicates percentage of live cells to total cells f Save to Data File record counting resultsin a table g Export stores all raw data assay parameters and graphsin an Excel file h Print send the current Counting Results to a printer i Done closes counting results window Note After clicking Done the counting results box can be redisplayed at any time by clicking on the View Report of Counting Results button 2009 Nexcelom Bioscience LLC Vision Manual 87 Tutorials Step 8 Review Imageson screen View Image AM Zoom In E Image F1 Image F2 Image Combined Counted A one of the fourca ptured images 1 2 3 or 4 one quadrant of a selected image FL fluorescence 1 image view F2 fluorescence 2 image view Combined check to display an overlay of Fl and Fl images Counted check to see counted cells outlined When both fluorescence channels are used check the Combined box to display an overlay of F1 and F2 captured images A FI B F2 2009 Nexcelom Bioscience LLC Assay 12 Splenocyte Concentration and Viability Using AO EB C Combined D Combined and Counted 2009 Nexcelom Bioscience LLC Vision Manual 89 Tutorials Modify Default Assay Parameters Vision default assay types are intended asguidelinesforsetting up new assays
70. t Assay Parameters for instructions on how to modify and save asa new assay Step 1 Stain sperm cells with propidium iodide following manufacturer s instructions Step 2 Select Assay 10 PI Sperm from the Assay SETUP drop down menu Cell Type sperm FL Assay 10 Imaging Mode Fluorescence Only Imaging mode Fluorescence Only Click on the yellow pencil on the nght side of Assay selection to view Assay Type Assay Name Assay 10 PI Sperm Assay is locked from editing Save as Mew Assay Type Special Cells Description Frog sperm cells stained with PI for total cell concentration Imaging Mode Fluorescence F1 Only Take BR with FL images Use BR Cell for F1 Count F1 Image Cell Type Sperm FL Assay 10 Description Edit Fluorophore PI Ex 525 nm 202 Fluorescent Exp 4900 0 meer Filter Set The Fluorescent Exposure time can be adjusted to obtain adequate signal within a range of PI concentrations since the signal strength is cell dependent Step 4 Click Preview Bnghtfield Image Step 5 Adjust focus if necessary Step 6 Click Count 76 2009 Nexcelom Bioscience LLC Assay 10 Sperm Cell Concentration Using Propidium lodide Staining Step 7 Review Counting Results and display data asneeded Counting Results Sample Sperm PI Dilution 2 0 Assay Assay 10_PI_Sperm Description Frog sperm cells stained with PI for total cell concentrat Cell Sperm_FL_Assay 10 Descriptio
71. tinguish and individually count cells from a cluster of cells Brightfield A standard imaging mode of the instrument using brightfield opticsfor viewing and counting total cells measuring cell szes and determining viability using trypan blue Huorescence The fluorescence imaging capabilities and modesof the instrument Used to detect fluorescence properties of cells Vision Duo hasone fluorescence channel while Vision Trio hastwo Bightfield Image displayed on screen after it hasbeen acquired from the brightifeld optics Image Huorescent Image displayed on screen after it has been acquired from the fluorescence optics Image F1 F2 F1 is the first fluorescence image F2 isthe second fluorescence image Threshold The limit to include orexclude a cell in a particular category counted not counted live dead fluorescence positive negative etc based on a particular property of the cell i e size fluorescence level bright center etc Count The command to initiate an image capture and analysis of a sample Speed Count The command to initiate an image capture and analysis of a sample in Speed Count mode ES Modify Assay or Cell Type parameters click to quickly access parameters from the main screen Generate Data and Reports click to view Counting Results box after initial pop up screen GH hasbeen cleared Sample Adjustment Calculator click to disolay the Sample Adjustment Calculator Useful for sample adjustment to get desire
72. to display an overay of Fl and Fl images Counted check to see counted cells outlined When both fluorescence channels are used check the Combined box to display an overlay of F1 and F2 captured images A Brightfield cell image B Counted cells 2009 Nexcelom Bioscience LLC Vision Manual 57 Tutorials C YFP positive cell image D Green outline indicates counted YFP positive cells 58 2009 Nexcelom Bioscience LLC Assay 06 White Blood Cell Concentration in Whole Blood with Acndine Orange Staining Assay 06 White Blood Cell Concentration in Whole Blood with Acridine Orange Staining Determining white blood cell counts in whole blood normally involvesa lysis procedure to eliminate red blood cells However WBCscan be counted in whole blood without lysis of RBCs by staining the blood sample with Acridine orange a nuclearstain that emits in the green range Aftertaking green fluorescent images of AO stained whole blood all fluorescent cells are counted and their concentration in whole blood isdetermined Brightfield images of the sample can be taken but are not used for WBC counting If RBC counting results are desired the original blood sample can be diluted approximately 1 1000 and counted using only brightfield images asin Assay 01 Note Assay parameters for assays that are included in the software are locked and can not be edited To customize this assay to suit your particularapplication please see Modify Def
73. ts produced using a combination of both images Fluorescence 1 F1 and Fluorescence 2 F2 use two different fluorescence imagesto produce counting results Trypan Blue Viability Check for trypan blue viability Use BR Cell for F1 Count Measure Fluorescence intensity only within cells located in Brightfield BR image F 1 Image Cell Type Cell Une Assay 03 Description Edit Fluorophore GFP Ex_475nm 101 Fluorescent Exp 2000 0 msec Filter Set Protocol Notes Browse Show Report Format Show F1 Image ContainsCell Type and Fluorescence parameters for the F1 image F2 Image Not shown Visible in Fluorescence 1 and Fluorescence 2 Imaging Mode contains Cell Type and Fluorescence parametersforthe F2 image Cell Type drop down menu to select cell parameters Edit edit selected cell type parameters Fluorophore User defined name of Fluorophore used in this assay Note 101 or 202 indicatesthe filter set that detects that particular fluorophore Fluorescent Exp Fluorescence exposure time in milliseconds Protocol Notes Link to any user defined file containing notes information about this particular assay Show Opens file loaded in Protocol Notes Browse find saved protocols 2009 Nexcelom Bioscience LLC Software Operation Overview son w Show Cell Count P lw Show Cell Mean Diameter Im lw Show Dilution jw Show Concentration lw For Fluorescent Positive Cells Use BrightField Cell
74. ularapplication please see Modify Default Assay Parameters for instructions on how to modify and save asa new assay Step 1 Trypsinize atta ched cells Step 2 Load 20uL in one side of the cell counting chamber and insert into instrument Step 3 Select Assay 7 05 YFP Transfection Rate from Assay SETUP drop down menu Imaging Mode Brightfield and Fluorescence Cell Type Cell Line_No decluster_Assay 05 is displayed Imaging Mode Brightfield and Fluorescence The brightfield imaging mode is used to measure total numberof cells and cell size The fluorescence imaging mode is used to measure fluorescence positive cells Both Cell Type and Imaging Mode are defined in the Assay parameters and can be edited from the main screen by clicking on the pencil icon Assay Type Assay Name Assay 05 YFP TransfechonRate Assay is locked from editing Save as New Assay Type Special Cells Description Cell line transfection rate measurement using YFP Imaging Mode Brightfield BR amp Fluorescence F1 Trypan Blue Viability M Use BR Cell for F1 Count Fi Image Cell Type cel Line_No deduster_Assay 05 Description Cell line cells not dumpy Edit Fluorophore YFP Ex 475 nm 101 Fluorescent Exp 2000 0 msec Filter Set Protocol Notes Browse Show Report Format Show m 52 2009 Nexcelom Bioscience LLC Assay 05 YFP Transfection Rate Step 4 Click Preview bnghteld Image
75. ustment calculator f Save to Data File record counting resultsto a data file g Export stores all raw data assay parameters and graphsin an Excel file h Print send cument counting resultsto printer i Done close counting results wind ow Note After clicking Done the counting results box can be redisplayed atany time by clicking on the View Report of Counting Results button 2009 Nexc elom Bioscience LLC Vision Manual 29 Tutorials d Show Size Distribution Cell Size Analysis Diameter File Name C Documents and Settings 4Il Users pplication Data Nexcelom_Vision5 diameters txt Cell Diameters Histogram Diameter um a Number of Cells Cell Diameter Histogram Percentage of Total Count ERE Ieee RAR IO IO CTT l Total Cell Count 1048 ezg Mean Diameter 12 0 Size Microns um 1 2 3 4 5 6 y 8 3 Percent 95 v Open Diameter File Multiple Save s Copy to Clipboard Cancel Print print cell size histogram Save As save cell diameter values to a data file Open Diameter File open an existing cell diameter file Multiple check to load and display multiple cell diameter files up to 5 Copy to Clipboard copy diameter data and histogram to Clipboard Thisdata can be pasted into a Word Excel or PowerPoint document OK Cancel 2009 Nexcelom Bioscience LLC Assay 01 Total Cell Concentration Measurement e Sample Adjustment Measured Concentration ce

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