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2 protocols using anti cd14 pe cy5

1

Multicolor Flow Cytometry Compensation Beads Protocol

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UltraComp eBeadsTM Compensation Beads (Thermofisher) were used to optimize fluorescence compensation settings for multi-color flow cytometric analysis at a Symphony flow cytometer. UltraComp eBeadsTM were stained with the following fluorochrome-labeled anti-human antibodies: anti-CD8–BUV805 (1:200, clone SK1), anti-CD4–BUV496 (1:50, clone SK3), anti-CD86–BUV737 (1:50, clone 2331 FUN-1), anti-CD141–BUV615-P (1:50, clone 1A4), anti-CD56–BUV563 (1:50, clone NCAM 16.2), anti-CD16–BUV395 (1:50, clone 3G8), anti-CD123–BB660-P (1:50, clone 7G3), anti-CD80–BB630 (1:50,clone L307.4), anti-CD21–BV785 (1:50, clone B-ly4), anti-CD27–BV750-P (1:40,clone L128), anti-BAFF-R–BV650 (1:50, clone 11C1), anti-CD94–BV605 (1:50, clone HP-3D9), anti-CD40–APC-R700 (1:50, clone 5C3) (all BD bioscience); anti-CD3–PerCP-Vio700 (1:50, clone REA613) (Miltenyi Biotec); anti-CD57–FITC (1:100, clone TB01), anti-CD14–PE-Cy5.5 (1:200, clone TuK4), fixable viability dye eFluor780 (1:1000) (all eBioscience); anti-CD24–BV711 (1:50, clone ML5), anti-CD19–BV510 (1:25, clone HIB19), anti-HLA-DR–BV570 (1:40, clone L243), anti-IgM–BV421 (1:100, clone MHM-88), anti-CD11c–APC (1:40, clone 3.9), anti-CD38–PE/Dazzle 594 (1:100, clone HB-7), anti-CD10–PE-Cy5 (1:50, clone HI10a), and anti-IgD–PE-Cy7 (1:100, clone IA6-2) (all BioLegend).
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2

Comprehensive Immunophenotyping of PBMC Subsets

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Frozen PBMCs from human healthy donors were processed as for the HS1 datasset and stained with live/dead marker and fluorochrome-conjugated antibodies against the following surface markers: anti-CD8-BUV805, anti-CD4-BUV496, anti-CD95-BUV737, anti-CD28-BB660-P, anti-ICOS-BB630, anti-CXCR3-BV785, anti-PD-1-BV750-P, anti-CXCR5-BV650, anti-CCR2-BV605, (all BD Biosciences); anti-CD3-PerCP-Vio700 (Mil-tenyi Biotec); anti-CD45RA-FITC, anti-CD14-PE-Cy5.5, fixable viability dye eFluor780 (all eBioscience); anti-CD25-BV711, anti-CD31-BV480, anti-HLA-DR-BV570, anti-CD127-BV421, anti-CCR4-PE/Dazzle 594, anti-CCR7-PE-Cy7 (all BioLegend).
Samples were stained for 60 min at 4
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