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What is Flow Cytometry ?

What is Flow Cytometry ?. Flow Cytometry. Introduction to Flow Cytometry. IGC Workshop. uic. Multicolor Flow Cytometry. Rui Gardner. IGC – April 28, 2010.

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What is Flow Cytometry ?

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  1. WhatisFlowCytometry? Flow Cytometry Introduction to Flow Cytometry IGC Workshop uic Multicolor FlowCytometry Rui Gardner IGC – April 28, 2010 AdaptedfromHoldenandTrotter (Winter 2006) “SelectingReagents for Multicolor FlowCytometry” BD Hotlinesnewsletter, 11: 31.-34

  2. Outline • KnowYourInstrument • OpticalLayout (lasers andfilters) • Choosingtherightfluorochromes • StainingIndex • Spillover • Compensation • ColorSpecificitiesand Tandem Dyes • Rules for Multicolor Analysis

  3. KnowYourInstrument

  4. KnowYourInstrument • ReagentSelectionstartswithInstrumentConfiguration • Lasers • Detectorsandrespectivefilters FACSCalibur FACScan CyAn ADP Analyzers FACSAria MoFlo CellSorters

  5. KnowYourInstrument(FACScan) 400 450 500 550 600 650 700 750 800 FACScanOpticalConfiguration TypicalFluorochromes 488 650LP 530/30 585/42 FL1 FL2 FL3 PE PI Cy3 GFP FITC Alexa488 CFSE PI PE-Cy5 PE-Cy7 PerCP PerCP-Cy5.5 7AAD PE-Alexa610 488 nm

  6. KnowYourInstrument(FACSCalibur) FACSCaliburOpticalConfiguration TypicalFluorochromes 400 450 500 550 600 650 700 750 800 400 450 500 550 600 650 700 750 800 488 670LP 530/30 585/42 FL1 FL2 FL3 488 nm PE PI Cy3 GFP FITC Alexa488 CFSE PI PE-Cy5 PE-Cy7 PerCP PerCP-Cy5.5 7AAD PE-Alexa610 633 661/16 FL4 APC Cy5 Alexa647 633 nm

  7. KnowYourInstrument(CyAn ADP) CyAn ADP Optical Configuration TypicalFluorochromes 405 450/50 530/40 400 450 500 550 600 650 700 750 800 400 450 500 550 600 650 700 750 800 400 450 500 550 600 650 700 750 800 Alexa 430 AmCyan Pacific Orange DAPI Alexa 405 PacificBlue Violet 1 (FL6) Violet 2 (FL7) 405 nm 488 530/40 575/25 613/20 680/30 750LP PI PE-TexasRed PE-Alexa 610 PE PI GFP FITC Alexa488 CFSE PI PE-Cy5 PerCP PerCP-Cy5.5 PE-Cy7 FL1 FL2 FL3 FL4 FL5 488 nm 642 665/20 750LP APC Cy5 Alexa647 APC-Cy7 APC-H7 Alexa 700* APC (FL8) APC-Cy7 (FL9) 642 nm

  8. KnowYourInstrument(FACSAria) FACSAriaOpticalConfiguration TypicalFluorochromes 407 450/40 530/30 400 450 500 550 600 650 700 750 800 400 450 500 550 600 650 700 750 800 400 450 500 550 600 650 700 750 800 Alexa 430 AmCyan Pacific Orange DAPI Alexa 405 PacificBlue 407 nm DAPI Alexa 430 488 530/30 585/42 616/23 695/40 780/60 PI PE-TexasRed PE-Alexa 610 PE PI GFP FITC Alexa488 CFSE PI PE-Cy5 PerCP PerCP-Cy5.5 PE-Cy7 488 nm FITC PE-TexasRed PE PerCP-Cy5.5 PE-Cy7 633 660/20 780/60 APC Cy5 Alexa647 APC-Cy7 APC-H7 Alexa 700* 633 nm APC APC-Cy7

  9. KnowYourInstrument(MoFlo) MoFloOpticalConfiguration #5 PE-Cy75 #6 795/50 APC PE-TxRed #4 645DLP 616/26 #7 670/40 PE #3 610DLP 585/40 H-Blue #8 FITC #2 555DLP 530/40 Red D405/30 H-Red mCherry SSC 95/5BS #9 670/30 488/10 616/26 #1 565 DCLP Yellow Blue UV

  10. ChoosingTheRightFluorochromes

  11. D W1 W2 Fluorochromes(StainIndex) BrightestFluorochrome = HighestStainIndex StainIndex (SI) =D/W

  12. Fluorochromes(StainIndex) Freshly isolated lymphocytes, stained with anti-human CD3 antibodies conjugated with various fluorochromes

  13. Fluorochromes(Choosethebrightest) StainIndexofvarious anti-CD4 fluorochromeconjugatesmeasuredon a BD LSR II

  14. Spillover (Minimize spillover) A singlefluorochromecanbedetectedin more thanonechannel Spectral Overlap Correcting overlap

  15. Compensation Compensationis a mathematicalsubtraction to correctspectraloverlap A488true = A488measured - % 1 A488true = A488measured - % PE true 0 % 5 % 10 % 15 % 20 % 30 % PE true = PE measured - % A488true Alexa488 http://www.drmr.com/compensation/index.html http://http://igc-wiki.igc.gulbenkian.pt/ PE

  16. ColorsandAntibody Specificities • (Reserve bright labels for dim antibodies) SingleStainControls SingleStainControls CD4-Alexa488 Fluorescence CD4-PE Fluorescence CD25-PE Fluorescence CD25-Alexa488 Fluorescence CD4-Alexa488 Fluorescence CD4-PE Fluorescence CD25-PE Fluorescence CD25-Alexa488 Fluorescence

  17. ColorsandAntibody Specificities • (Avoid spillover of bright cells into detectors of dim signals) SingleStainControls Sample CD4-Cy5 Fluorescence CD4-Cy5 Fluorescence CD4-Alexa488 Fluorescence CD4-Alexa488 Fluorescence CD25-PE Fluorescence CD25-PE Fluorescence CD25-PE Fluorescence CD25-PE Fluorescence CD4-Alexa488 Fluorescence CD25-PE Fluorescence

  18. Tandem Dyes Watchout for degradation TIME APC- 30% PE-Cy5 APC- 40% PE-Cy5 APC-50% PE-Cy5 PE-Cy5 Fluorescence PE-Cy5 Fluorescence PE-Cy5 Fluorescence APC Fluorescence APC Fluorescence APC Fluorescence PE-Cy5 PE-Cy7 APC-Cy7 APC-H7

  19. Rules for choosingtherightfluorochromes

  20. “Rules” for selecting Multicolor Panel • takenfrom BD Biosciences Rule 1: Choosethebrightestsetoffluorochromes for your particular instrumentconfiguration. Rule 2: Choosefluorochromesso as to minimize thepotential for spillover. Rule 3: Reserve thebrightestfluorochromes for “dim” antibodies, and vice versa. Rule 4: Avoidspilloverfrombrightcellpopulationsintodetectorsrequiringhighsensitivity for thosepopulations. Rule 5: Takesteps to avoid tandem dyedegradation, andconsideritsimpactuponresults.

  21. Recommended Multicor Panel Fluorochromechoices for 5 or more colors(Recommendedby BD)

  22. Recommended Multicor Panel Fluorochromechoices for 5 or more colors(Recommendedby IGC)

  23. WhatisFlowCytometry? Flow Cytometry Introduction to Flow Cytometry IGC Workshop uic Multicolor FlowCytometry(end) Rui Gardner IGC – April 28, 2010 AdaptedfromHoldenandTrotter (Winter 2006) “SelectingReagents for Multicolor FlowCytometry” BD Hotlinesnewsletter, 11: 31.-34

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