1. Building a Panel Select markers Select conjugates Method to build panel performance criteria

2. Select markers Endpoints – essential measures for study (cytokines) Anchor gates – necessary to identify cell populations expressing endpoint values Markers to improve sensitivity – viability gate Ancillary markers – everything else, in order of priority Building a Panel

3. Select conjugates Instrument configuration Lasers Filters # parameters Availability Commercial Custom In-house Experience Building a Panel

4. Staining Index Varies by Instrument

5. The basics of selecting fluorophores Choose the brightest fluorophores that can be used on your instrument Use brightest fluorophore for your least expressed protein Use the dimmest fluorophore for your most highly expressed protein (CD45, CD8, CD3) Avoid spillover from bright cell populations into detectors requiring high sensitvity Spectral viewer tools Use tandems with caution Essential for large multicolor panels Susceptible to degradation by light exposure or fixation Prevent light exposure Use appropriate fixation buffers and protocols Avoid acidic buffer conditions with FITC labeled samples because FITC is sensitive to low pH Avoid exposing samples and reagents to bright light Avoid incubating cells in fixative for extended periods of time, as this may affect fluorescence, particularly of tandems dyes Acquire samples with 6 hours of staining

6. Method to build panel – performance criteria Titrations May require stimulation to obtain maximum expression Saturation required for fluorescence quantitation Combining markers - spillover Empirical (MoFUN) Using single-stained titration files (OMIP 006) plus testing final panel options Instrument Controls Assay-specific target channels (Perfetto) Compensation beads - as bright as or brighter than cells Building a Panel

7. OMIP 006 Murdoch et. al. Cytometry A. 2012 Apr;81(4):281-3

8. OMIP 006 Murdoch et. al. Cytometry A. 2012 Apr;81(4):281-3

9. OMIP 006 Murdoch et. al. Cytometry A. 2012 Apr;81(4):281-3

10. OMIP 006 Murdoch et. al. Cytometry A. 2012 Apr;81(4):281-3

11. OMIP 006 Murdoch et. al. Cytometry A. 2012 Apr;81(4):281-3

12. OMIP 006 Murdoch et. al. Cytometry A. 2012 Apr;81(4):281-3

13. OMIP 006 Murdoch et. al. Cytometry A. 2012 Apr;81(4):281-3

14. OMIP 006 Murdoch et. al. Cytometry A. 2012 Apr;81(4):281-3

15. OMIP 006 Murdoch et. al. Cytometry A. 2012 Apr;81(4):281-3

16. OMIP 006 Murdoch et. al. Cytometry A. 2012 Apr;81(4):281-3

17. Tools & Resources OMIP – Optimized Multi-color Immunophenotyping Panel, Cytometry special publication format Fluorish Chromocyte (Flow Cytometry Network) Beckman Coulter – Antibody Panel Creator BD Biosciences – Multicolor Flow Cytometry BioLegend – Multicolor Panel Selector