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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 03, 2013.

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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 03, 2013 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 HyperCyt FACSAria MoFlo CellSorters LSR Fortessa

  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. KnowYour Instrument (LSR Fortessa) LSR Fortessa Optical Configuration 561 nm (YG) 488 nm (Blue) 442 nm (BV)

  11. KnowYour Instrument (LSR Fortessa) PE-Cy5, PE-A647 mPlum PE RFP, DsRed dTomato mOrange YG:561nm YELLOW GREEN (561 nm) 670/40 590/30 650LP 740LP 610LP 780/60 630/30 PE-Cy7 PE-TexasRed PI mCherry mRaspberry mplum 630/75 or 590LP in position A For mCherry detection only

  12. KnowYour Instrument (LSR Fortessa) SSC (BV) CFP BLUE VIOLET (442 nm) 445/15 577LP 455LP 470/20 442 nm (BV)

  13. KnowYour Instrument (LSR Fortessa) PE-Cy5 Blue:488nm SSC BLUE (488 nm) 690/40 488/10 655LP 740LP 502LP 780/60 530/30 PE-Cy7 FITC Alexa 488 GFP YFP

  14. KnowYour Instrument (LSR Fortessa) YFP Blue:488nm SSC BLUE (488 nm) 540/30 488/10 525LP 740LP 502LP 780/60 510/20 PE-Cy7 GFP Measure GFP and YFP simultaneously

  15. ChoosingTheRightFluorochromes AdaptedfromHoldenandTrotter (Winter 2006) “SelectingReagents for Multicolor FlowCytometry” BD Hotlinesnewsletter, 11: 31.-34

  16. Rule 1 Choose the Brightest Fluorochromes

  17. D W1 W2 Fluorochromes(StainIndex) BrightestFluorochrome = HighestStainIndex MFIPOSITIVE ̶ MFINEGATIVE Stain Index = 2 × rSDNEGATIVE StainIndex (SI) =D/W

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

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

  20. Rule 2 Minimize Potential Spillover

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

  22. 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/ PE Roederer, M. 2002. Compensation in Flow Cytometry. Current Protocols in Cytometry. 1.14.1–1.14.20

  23. Spillover (Minimize spillover) Slide taken from presentation: Mario Roederer, “Compensation: Basic Principles”, Monday, June 20, 2011

  24. Rule 3 • Reserve brightest fluorochromes for “dim” antibodies and vice-versa

  25. 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

  26. Rule 4 • Avoid spillover from bright populations into detectors requiring high sensitivity

  27. 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

  28. Rule 5 • Take steps to avoid tandem dye degradation

  29. 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

  30. “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.

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

  32. Recommended Multicor Panel Fluorochromechoices for 5 or more colors(Recommendedby IGC) Completely Outdated!

  33. WhatisFlowCytometry? Flow Cytometry Introduction to Flow Cytometry IGC Workshop uic Multicolor FlowCytometry(end) Rui Gardner IGC – Aprli 03, 2013

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