• Document: Best practices in panel design to optimize the isolation of cells of interest
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Sort Best practices in panel design to optimize the isolation of cells of interest For Research Use Only. Not for use in diagnostic or therapeutic procedures. Alexa Fluor® is a registered trademark of Life Technologies Corporation. Cy™ is a trademark of GE Healthcare. Cy™ dyes are subject to proprietary rights of GE Healthcare and Carnegie Mellon University and are made and sold under license from GE Healthcare only for research and in vitro diagnostic use. Any other use requires a commercial sublicense from GE Healthcare, 800 Centennial Avenue, Piscataway, NJ 08855-1327, USA. Trademarks are the property of their respective owners. © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company. The BD Horizon™ Global Tour | 1 23-18846-00 Cell sorting Cell sorting: isolation of a population of interest for downstream analysis • Cell enrichment • Cell transplantation • Downstream functional and genomic analysis Goal: to obtain a pure population with maximum yield The BD Horizon™ Global Tour | 2 Increasing resolution for cell sorting • Clear resolution of a population of interest is critical for an optimal sort. • How to increase resolution in a sorting setting? – Eliminate the impact of unwanted cells – Increase the ability to visualize from population of interests. The BD Horizon™ Global Tour | 3 Increasing resolution for cell sorting • Clear resolution of a population of interest is critical for an optimal sort. PE PE-CF594 BV421 Alexa Fluor® 647 CD3 PerCP-Cy™5.5 Alexa Fluor® V450 FITC 700 CD197 (CCR7) • How to increase resolution in a sorting setting? – Eliminate the impact of unwanted cells. – Increase the ability to visualize population of interests. The BD Horizon™ Global Tour | 4 Considerations for cell sorting Cell analysis Cell sorting • Fluorochromes • Fluorochromes • Biology • Biology • Instrument • Instrument setup setup • Sample preparation • Gating strategy The BD Horizon™ Global Tour | 5 Considerations for cell sorting: overview • Experiment setup • Panel design – Sample preparation – Dead cell exclusion – Instrument settings – Lineage exclusion/depletion • Gating strategy – Antibody titration – Histogram vs plots – Fluorochrome choice – Biexponential scale – Doublet discrimination – Data display The BD Horizon™ Global Tour | 6 Considerations for cell sorting Sample preparation, instrument setup The BD Horizon™ Global Tour | 7 Sample preparation • Cell dissociation/detachment • Cell resuspension buffer • Staining volume and antibody concentration • Cell sorting buffer and cell density • Optional use of DNAse • Temperature, pH • Sample collection: – Cell collection buffer (cell culture, transplantation, genomic analysis) – Temperature, pH The BD Horizon™ Global Tour | 8 Instrument settings • Nozzle size/sheath pressure – 70 µm/70 psi for lymphocytes – 100 µm/20 psi for larger and/or fragile cells • Event rate (number of events/second) – Low speed for higher sorting efficiency • Sort setup – Bulk sorting – Purity vs yield – Single cells • Laser alignment • Drop delay The BD Horizon™ Global Tour | 9 Instrument settings: speed vs yield vs purity • Influenced by: – Drops per second, events per second, sort “mask” and target population frequency 100 90 Speed 80 70 Percent Yield 60 50 40 30 20 10 0 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 Purity Yield Events/second (x 1000) • Good rule of thumb: – Maximum recommended event rate = drops per second / 5 The BD Horizon™ Global Tour | 10 Considerations for cell sorting Gating strategy Histogram vs plots, biexponential scale, doublet discrimination, data display The BD Horizon™ Global Tour | 11 Different options for data disp

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