• Document: Beyond Colorful Science. A Practical Approach to Setting Up Multicolor Panels
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Beyond Colorful Science A Practical Approach to Setting Up Multicolor Panels Dr. Andreas Böhmler ISCT Paris∙ April 2014 Layout: Multicolor Panel Design • Identify the pitfalls • Have a look at issues with spectral overlap • How to design a robust multicolor panel Dr. Andreas Böhmler Beckman Coulter Flow Cytometry EMEAI Version 1.0 / 2014 ISCT 2014 Flow Cytometry and Cell Analysis Workshop Panel Design Objectives • High sensitivity for dim antigens, independent of their expression pattern (discrete or modulated). • Clear signal separation from unspecific background. • Robust spillover patterns. Dr. Andreas Böhmler Beckman Coulter Flow Cytometry EMEAI Version 1.0 / 2014 ISCT 2014 Flow Cytometry and Cell Analysis Workshop Panel Design Toolbox 6 rules based on • Dye performance characteristics • Spectral overlap • Co-expression patterns Dr. Andreas Böhmler Beckman Coulter Flow Cytometry EMEAI Version 1.0 / 2014 ISCT 2014 Flow Cytometry and Cell Analysis Workshop Panel Design Toolbox 6 rules based on • Dye performance characteristics • Spectral overlap • Co-expression patterns Dr. Andreas Böhmler Beckman Coulter Flow Cytometry EMEAI Version 1.0 / 2014 ISCT 2014 Flow Cytometry and Cell Analysis Workshop Dim Marker, Bright Fluorochrome? Dr. Andreas Böhmler Beckman Coulter Flow Cytometry EMEAI Version 1.0 / 2014 ISCT 2014 Flow Cytometry and Cell Analysis Workshop Dye ‚Brightness‘ The requirement for brightness depends on the antigen density and expression characteristics. Dr. Andreas Böhmler Beckman Coulter Flow Cytometry EMEAI Version 1.0 / 2014 ISCT 2014 Flow Cytometry and Cell Analysis Workshop Dye Spillover ECD 488nm KrO Brightness is not always better but an ambiguous dye property as it may involve strong spillover and data spreading. Dr. Andreas Böhmler Beckman Coulter Flow Cytometry EMEAI Version 1.0 / 2014 ISCT 2014 Flow Cytometry and Cell Analysis Workshop Spectral Consistency APCA750 (CD27, CD45RA) PC5.5 (CD25, CD56, CD19) APCA700 (CD25, CD16) Overlays of separate stainings using a variety of specificities with identical conjugation Multicolor cytometry requires first class tandem dyes with high spectral consistency Dr. Andreas Böhmler Beckman Coulter Flow Cytometry EMEAI Version 1.0 / 2014 ISCT 2014 Flow Cytometry and Cell Analysis Workshop Panel Design Toolbox 6 rules based on • Dye performance characteristics • Spectral overlap • Co-expression patterns Dr. Andreas Böhmler Beckman Coulter Flow Cytometry EMEAI Version 1.0 / 2014 ISCT 2014 Flow Cytometry and Cell Analysis Workshop Ideal World: No Spectral Overlap 488nm APC 638nm Pacific Blue 405 nm Dr. Andreas Böhmler Beckman Coulter Flow Cytometry EMEAI Version 1.0 / 2014 ISCT 2014 Flow Cytometry and Cell Analysis Workshop Impact of Spectral Overlap HLA-DR - PE Loss of sensitivity CD45-FITC CD45-FITC HLA-DR - PE CD45-Pac Blue CD45-Pac Blue Dr. Andreas Böhmler Beck

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