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Building a Panel

Building a Panel. Select markers Select conjugates Method to build panel performance criteria. Building a Panel. Select markers Endpoints – essential measures for study (cytokines) Anchor gates – necessary to identify cell populations expressing endpoint values

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Building a Panel

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

  2. Building a Panel 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

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

  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. Building a Panel 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

  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

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