1. What is Flow Cytometry? Flow Cytometry uic Introduction to Flow Cytometry IGC Workshop Multicolor Flow Cytometry IGC – April 28, 2010 Adapted from Holden and Trotter (Winter 2006) “Selecting Reagents for Multicolor Flow Cytometry” BD Hotlines newsletter, 11: 31.-34

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

3. Know Your Instrument

4. Reagent Selection starts with Instrument Configuration Lasers Detectors and respective filters Analyzers Cell Sorters FACSCalibur FACScan MoFlo FACSAria CyAn ADP

5. Know Your Instrument (FACScan) FACScan Optical Configuration 400450500550600650700750800 488 530/30585/42 650LP FL1FL2FL3 GFP FITC Alexa488 CFSE PE PI Cy3 PI PE-Cy5 PE-Cy7 PerCP PerCP-Cy5.5 7AAD PE-Alexa610 Typical Fluorochromes 488 nm

6. Know Your Instrument (FACSCalibur) FACSCalibur Optical Configuration Typical Fluorochromes 400450500550600650700750800 488 530/30585/42 670LP FL1FL2FL3 488 nm 633 nm 400450500550600650700750800 633 661/16 FL4 APC Cy5 Alexa647 GFP FITC Alexa488 CFSE PE PI Cy3 PI PE-Cy5 PE-Cy7 PerCP PerCP-Cy5.5 7AAD PE-Alexa610

7. Know Your Instrument (CyAn ADP) CyAn ADP Optical Configuration GFP FITC Alexa488 CFSE PE PI PI PE-Cy5 PerCP PerCP-Cy5.5 Typical Fluorochromes 405 nm 642 nm APC Cy5 Alexa647 400450500550600650700750800 488 nm 405 450/50 400450500550600650700750800400450500550600650700750800 488 642 Alexa 430 AmCyan Pacific Orange DAPI Alexa 405 Pacific Blue PI PE-Texas Red PE-Alexa 610 PE-Cy7 APC-Cy7 APC-H7 Alexa 700* 530/40 575/25613/20680/30750LP 665/20 FL1FL2FL3FL4FL5 APC (FL8) APC-Cy7 (FL9) Violet 1 (FL6) Violet 2 (FL7)

8. Know Your Instrument (FACSAria) FACSAria Optical Configuration GFP FITC Alexa488 CFSE PE PI PI PE-Cy5 PerCP PerCP-Cy5.5 Typical Fluorochromes 407 nm 633 nm APC Cy5 Alexa647 400450500550600650700750800 488 nm 407 450/40 DAPI Alexa 430 400450500550600650700750800 FITC PE PE-Texas Red PerCP-Cy5.5 PE-Cy7 400450500550600650700750800 APC APC-Cy7 488 633 Alexa 430 AmCyan Pacific Orange DAPI Alexa 405 Pacific Blue PI PE-Texas Red PE-Alexa 610 PE-Cy7 APC-Cy7 APC-H7 Alexa 700* 530/30 585/42616/23695/40780/60 660/20

9. 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 Know Your Instrument (MoFlo) MoFlo Optical Configuration H-Blue H-Red UV 670/30 565 DCLP D405/30 Yellow mCherry 616/26

10. Choosing The Right Fluorochromes

11. Fluorochromes (Stain Index) Brightest Fluorochrome = Highest Stain Index W2 W1 D Stain Index (SI) =D/W

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

13. Fluorochromes (Choose the brightest) Stain Index of various anti-CD4 fluorochrome conjugates measured on a BD LSR II

14. Spillover (Minimize spillover) A single fluorochrome can be detected in more than one channel Spectral OverlapCorrecting overlap

15. A488 true = A488 measured - % PE true PE true = PE measured - % A488 true 0 %5 %10 %15 %20 %30 % Compensation Compensation is a mathematical subtraction to correct spectral overlap http://www.drmr.com/compensation/index.html http://http://igc-wiki.igc.gulbenkian.pt/ Alexa488 PE A488 true = A488 measured - % 1

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

17. Sample CD25-PE Fluorescence CD4-Alexa488 Fluorescence Single Stain Controls CD4-Alexa488 Fluorescence CD25-PE Fluorescence Colors and Antibody Specificities (Avoid spillover of bright cells into detectors of dim signals) CD4-Cy5 Fluorescence CD25-PE Fluorescence

18. Tandem Dyes Watch out for degradation APC Fluorescence PE-Cy5 Fluorescence TIME PE-Cy5 PE-Cy7APC-Cy7APC-H7 APC- 30% PE-Cy5APC- 40% PE-Cy5APC-50% PE-Cy5

19. Rules for choosing the right fluorochromes

20. “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.

21. Recommended Multicor Panel 5-color6-color8-color10-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 Fluorochrome choices for 5 or more colors (Recommended by BD)

22. Recommended Multicor Panel 5-color6-color8-color10-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 AmCyan Pacific Blue Fluorochrome choices for 5 or more colors (Recommended by IGC)

23. What is Flow Cytometry? Flow Cytometry uic Introduction to Flow Cytometry IGC Workshop Multicolor Flow Cytometry (end) IGC – April 28, 2010 Adapted from Holden and Trotter (Winter 2006) “Selecting Reagents for Multicolor Flow Cytometry” BD Hotlines newsletter, 11: 31.-34