1. Design and optimization of multicolor panels Holden T. Maecker

2. Outline Choosing fluorochrome combinations and filter sets Matching antibody specificities with fluorochromes Controls and standardization

3. Outline Choosing fluorochrome combinations and filter sets Choose bright fluorochromes Minimize spillover between detectors

4. “Bright” = good resolution sensitivity W2 W1 D Where D = difference between positive and negative peak medians, and W = 2 x rSD (robust standard deviation)

5. Various fluorochromes-stain index ReagentCloneFilterStain Index PERPA-T4585/40356.3 Alexa 647RPA-T4660/20313.1 APCRPA-T4660/20279.2 PE-Cy7RPA-T4780/60278.5 PE-Cy5RPA-T4695/40222.1 PerCP-Cy5.5Leu-3a695/4092.7 PE-Alexa 610RPA-T4610/2080.4 Alexa 488RPA-T4530/3075.4 FITCRPA-T4530/3068.9 PerCPLeu-3a695/4064.4 APC-Cy7RPA-T47801/6042.2 Alexa 700RPA-T4720/4539.9 Pacific BlueRPA-T4440/4022.5 AmCyanRPA-T4525/5020.2

6. Spillover affects resolution sensitivity FITC background contributes noise to PE measurement www.bdbiosciences.com/spectra

7. Choices for 6-, 8-, 10-, and more colors 6-color8-color10-colorAdditional FITC or Alexa 488 PE PE-Texas Red or PE-Alexa 610 PerCP-Cy5.5 PE-Cy7 APC or Alexa 647 Alexa 680 or 700 APC-Cy7 AmCyan Pacific Blue Q-dot 655, 705…

8. Outline Choosing fluorochrome combinations and filter sets Matching antibody specificities with fluorochromes Controls and standardization

9. Fluorochrome selection considerations “Bright” antibodies go on “dim” fluorochromes Avoid spillover from bright cell populations into detectors requiring high sensitivity Take special care with tandem dyes

10. uncompensated compensated FITC mean fluorescencePE mean fluorescence---------------------------- negativepositivenegativepositive ------------------------------------------ uncompensated12335411841648 compensated1233564134136 data spread Data spread of fluorescent signals HT Maecker, T Frey, LE Nomura, J Trotter: Selecting fluorochrome conjugates for maximum sensitivity. Cytometry A 2004, 62:169-73.

11. Spillover affects resolution sensitivity Without CD45 AmCyan: With CD45 AmCyan: CD19 FITC Note that this is only an issue when the two markers (CD45 and CD19) are co-expressed on the same cell population.

12. Special requirements of tandem dyes Compensation requirements for tandem dye conjugates can vary, even between two experiments with the same antibody Require experiment-specific compensation Certain tandem dye conjugates (APC-Cy7, PE-Cy7) can degrade with exposure to light, elevated temperature, and fixation Minimize exposure to these conditions Use BD Stabilizing Fixative for final fixation

13. False positives due to tandem degradation A. False positives in APC channel reduced in absence of APC-Cy7 False positives in PE channel remain Gating schemeCD8 APC-Cy7+ cellsCD4 PE-Cy7+ cells B. With CD8 APC-Cy7 and CD4 PE-Cy7: Without CD8 APC-Cy7:

14. New tandems will be more stable APC-H7 as a replacement for APC-Cy7: Comparison of Sample Stability (in BD Stabilizing Fixative at RT) 0 50 100 150 200 250 0124682448 Hours of light exposure % Spillover CD4 APC-H7 CD8 APC-H7 CD4 APC-Cy7 CD8 APC-Cy7

15. Outline Choosing fluorochrome combinations and filter sets Matching antibody specificities with fluorochromes Controls and standardization

16. Types of controls Instrument setup controls PMT voltage settings Compensation (per experiment) Gating controls Isotype controls Fluorescence-minus-one (FMO) controls Biological controls Unstimulated samples Healthy donors

17. Comparison of gating controls

18. Standardization using lyophilized reagents Lyophilization provides increased stability, even at room temperature or 37 o C One batch of reagents can be used for an entire longitudinal study Pre-configured plates can avoid errors of reagent addition Complex experiments (multiple stimuli, multiple polychromatic staining cocktails) become easier Lyophilized cell controls can provide run-to- run standardization

19. Conclusions Polychromatic flow cytometry is not impossible Select fluorochromes for brightness and least spillover Optimize antibody panels by taking into account reagent brightness and data spread Stabilize longitudinal experiments with proper QC Some solutions that can help Lyophilized reagent plates Stabilizing fixative Beads for calibration and compensation

20. References Maecker, H. T., Frey, T., Nomura, L. E., and Trotter, J. (2004).Selecting fluorochrome conjugates for maximum sensitivity. Cytometry A 62, 169. Maecker, H. T., and Trotter, J. (2006).Flow cytometry controls, instrument setup, and the determination of positivity. Cytometry A 69, 1037.

21. Acknowledgements Laurel Nomura Margaret Inokuma Maria Suni Smita Ghanekar Daiva Gladding Jack Dunne Skip Maino Joe Trotter Dennis Sasaki Marina Gever