Following cell staining, samples were acquired in a BD LSR Fortessa SORP cytometer. The FACS tubes were acquired completely. Spectral overlap compensation between all channels was done automatically by the BD FACSDiva software v8.0.1 (BD Biosciences, Franklin Lakes, USA, New Jersey) using single-color controls. Comparable day-to-day performance of the cytometer was ascertained by running CS&T calibration beads (BD) weekly. In addition, for standardization of instrument settings longitudinally, an 8-peak Rainbow Compensation Particles Set (BD) was used prior to every experiment, and photomultiplier tubes (PMTs) voltages were adjusted if needed. Flow cytometry data were analyzed using FlowJo v10.8.1 (TreeStar, Portland, USA, OR). The FlowJo boolean gating tool was used to create the co-expression profiles.
8 peak rainbow compensation particles set
Manufactured by BD
The 8-peak Rainbow Compensation Particles Set is a laboratory equipment designed to facilitate the calibration and optimization of flow cytometry instruments. The set contains a mixture of fluorescently labeled particles with distinct emission spectra, enabling users to establish compensation settings and verify the performance of their flow cytometry system.
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2 protocols using 8 peak rainbow compensation particles set
1
Multicolor Flow Cytometry Standardization
2
Multicolor Flow Cytometry Analysis
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Fresh whole blood (100 μL) was stained with monoclonal, fluorescent-labelled antibodies (Supplementary Tables 1 and 2 ). Cells were incubated for 20 minutes at room temperature in the dark and lysed with FACS Lysing Buffer (BD Becton Dickinson), according to the manufacturer's instructions. After a washing step with phosphate-buffered saline plus 1% fetal bovine serum, pellets were resuspended in 300μL of Running Buffer (Miltenyi Biotec). before acquisition within 1–4 hours. Samples were acquired in a BD LSR Fortessa SORP cytometer. Spectral overlap compensation between all channels was done automatically by the BD FACSDiva software (BD Biosciences), using single-color controls. For standardization of instrument settings longitudinally, an 8-peak Rainbow Compensation Particles Set (BD) was used before every experiment, and photomultiplier tubes voltages were adjusted if needed. Flow cytometric data were analyzed using FlowJo software, version 10.8.1 (TreeStar). For coexpression patterns, the FlowJo Boolean tool was used (Supplementary Figure 1 ).
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