Flow Cytometry What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Flow Cytometry uic Multicolor Flow Cytometry Rui Gardner IGC – April 03, 2013 Adapted from Holden and Trotter (Winter 2006) “Selecting Reagents for Multicolor Flow Cytometry” BD Hotlines newsletter, 11: 31.-34

Outline Know Your Instrument Choosing the right fluorochromes Optical Layout (lasers and filters) Choosing the right fluorochromes Staining Index Spillover Compensation Color Specificities and Tandem Dyes Rules for Multicolor Analysis

Know Your Instrument

Know Your Instrument Reagent Selection starts with Instrument Configuration Lasers Detectors and respective filters FACScan FACSCalibur CyAn ADP Analyzers HyperCyt FACSAria MoFlo Cell Sorters LSR Fortessa

Know Your Instrument (FACScan) FACScan Optical Configuration Typical Fluorochromes 488 400 450 500 550 600 650 700 750 800 530/30 585/42 650LP FL1 FL2 FL3 GFP FITC Alexa488 CFSE PE PI Cy3 PI PE-Cy5 PE-Cy7 PerCP PerCP-Cy5.5 7AAD PE-Alexa610 488 nm

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

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

Know Your Instrument (FACSAria) FACSAria Optical Configuration Typical Fluorochromes 407 450/40 530/30 400 450 500 550 600 650 700 750 800 DAPI Alexa 405 Pacific Blue Alexa 430 AmCyan Pacific Orange 407 nm DAPI Alexa 430 488 530/30 585/42 616/23 695/40 780/60 400 450 500 550 600 650 700 750 800 GFP FITC Alexa488 CFSE PE PI PI PE-Texas Red PE-Alexa 610 PI PE-Cy5 PerCP PerCP-Cy5.5 PE-Cy7 488 nm FITC PE PE-Texas Red PerCP-Cy5.5 PE-Cy7 633 660/20 780/60 400 450 500 550 600 650 700 750 800 APC Cy5 Alexa647 APC-Cy7 APC-H7 Alexa 700* 633 nm APC APC-Cy7

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

Know Your Instrument (LSR Fortessa) LSR Fortessa Optical Configuration 488 nm (Blue) 561 nm (YG) 442 nm (BV)

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

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

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

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

Choosing The Right Fluorochromes Adapted from Holden and Trotter (Winter 2006) “Selecting Reagents for Multicolor Flow Cytometry” BD Hotlines newsletter, 11: 31.-34

Choose the Brightest Fluorochromes Rule 1 Choose the Brightest Fluorochromes

Fluorochromes (Stain Index) Brightest Fluorochrome = Highest Stain Index D W2 W1 MFIPOSITIVE MFINEGATIVE ̶ 2 × rSDNEGATIVE Stain Index = Stain Index (SI) =D/W

Fluorochromes (Stain Index) Freshly isolated lymphocytes, stained with anti-human CD3 antibodies conjugated with various fluorochromes

Fluorochromes (Choose the brightest) Stain Index of various anti-CD4 fluorochrome conjugates measured on a BD LSR II Reagent Clone Filter Stain Index PE RPA-T4 585/40 356.3 Alexa 647 660/20 313.1 APC 279.2 PE-Cy7 780/60 278.5 PE-Cy5 695/40 222.1 PerCP-Cy5.5 Leu-3a 92.7 PE-Alexa 610 610/20 80.4 Alexa 488 530/30 75.4 FITC 68.9 PerCP 64.4 APC-Cy7 7801/60 42.2 Alexa 700 720/45 39.9 Pacific Blue 440/40 22.5 AmCyan 525/50 20.2 However stain indexes are calculated with reagents run on their own, and not part of a cocktail

Minimize Potential Spillover Rule 2 Minimize Potential Spillover

Spillover (Minimize spillover) A single fluorochrome can be detected in more than one channel Spectral Overlap Correcting spillover

Compensation Compensation is a mathematical subtraction to correct spectral overlap A488true = A488measured - % PE true PE true = PE measured - % A488true A488true = A488measured - % 1 20 % 30 % 15 % 0 % 5 % 10 % Alexa488 PE http://www.drmr.com/compensation/ Roederer, M. 2002. Compensation in Flow Cytometry. Current Protocols in Cytometry. 1.14.1–1.14.20

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

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

Colors and Antibody Specificities (Reserve bright labels for dim antibodies) CD4-PE Fluorescence CD25-Alexa488 Fluorescence Single Stain Controls Single Stain Controls CD25-PE Fluorescence CD4-Alexa488 Fluorescence

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

Colors and Antibody Specificities (Avoid spillover of bright cells into detectors of dim signals) Single Stain Controls Sample CD25-PE Fluorescence CD4-Alexa488 Fluorescence CD4-Cy5 Fluorescence CD25-PE Fluorescence CD4-Alexa488 Fluorescence CD25-PE Fluorescence {{1000-200,2000},{5000-200,2000}}, PE vs A488 with PEtrue=PEmeas-10%A488True CD4-Alexa488 Fluorescence CD25-PE Fluorescence

Take steps to avoid tandem dye degradation Rule 5 Take steps to avoid tandem dye degradation

Tandem Dyes Watch out for degradation TIME PE-Cy5 PE-Cy7 APC-Cy7 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

“Rules” for selecting Multicolor Panel taken from BD Biosciences Rule 1: Choose the brightest set of fluorochromes for your particular instrument configuration. Rule 2: Choose fluorochromes so as to minimize the potential for spillover. Rule 3: Reserve the brightest fluorochromes for “dim” antibodies, and vice versa. Rule 4: Avoid spillover from bright cell populations into detectors requiring high sensitivity for those populations. Rule 5: Take steps to avoid tandem dye degradation, and consider its impact upon results.

Recommended Multicor Panel Fluorochrome choices for 5 or more colors (Recommended by BD) 5-color 6-color 8-color 10-color FITC or Alexa 488 PE PE-Texas Red or PE-Alexa 610 PerCP-Cy5.5 PE-Cy7 APC , Alexa 647 or Cy5 Alexa 700 APC-Cy7 AmCyan Pacific Blue

Completely Outdated! Recommended Multicor Panel Fluorochrome choices for 5 or more colors (Recommended by IGC) 5-color 6-color 8-color 10-color FITC or Alexa 488 PE PE-Texas Red or PE-Alexa 610 PerCP-Cy5.5 or PerCP PE-Cy7 APC , Alexa 647 or Cy5 Alexa 700 APC-Cy7 Pacific Orange Pacific Blue Completely Outdated!

Flow Cytometry What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Flow Cytometry uic Multicolor Flow Cytometry (end) Rui Gardner IGC – Aprli 03, 2013