Anti human cd19 efluor450

Blood was collected in EDTA tubes, and 100 μl was incubated with anti-human CD19-eFluor450 (eBioscience, San Diego, CA, USA), CD24-fluorescein isothiocyanate (FITC) (BD biosciences, Franklin Lakes, NJ, USA), CD27-APC-eFluor780 (eBioscience), CD38-PeCy5 (eBioscience), CD5-PerCp-Cy5.5 (Biolegend, San Diego, CA, USA) or the corresponding isotype controls. After 15 minutes cells were treated with fluorescence-activated cell sorting (FACS) Lysing solution (BD Biosciences). Samples were measured using an LSR-II flow cytometer (BD biosciences) and data were analyzed using Kaluza 1.2 flow analysis software (Beckman Coulter, Brea, CA, USA). B cells were divided into transitional, memory, naive, CD24^highCD38^high and CD24^highCD27+ B cells as described previously [14 (link)]. CD5+ B cells were gated on an isotype control.

EDTA venous blood was obtained from patients and HCs and immediately washed twice in PBS with 1% BSA (wash buffer). Next, 100 μl blood was incubated with anti-human CD19-eFluor450, CD27-APC-eFluor780, CD38-PE-Cy7 (eBioscience, San Diego, CA, USA), CD24-FITC, IgM-APC, IgD-PE (BD Biosciences, San Jose, CA, USA) or corresponding isotype controls for 15 minutes and treated with 10x FACS Lysing solution (BD Biosciences) for 10 minutes. After washing, samples were acquired on a FACS LSR-II flow cytometer (BD Biosciences). At least 200,000 events were measured and plotted using Kaluza v1.5a flow analysis software (Beckman Coulter, Brea, CA, USA) or FlowJo v10 analysis software (Treestar, Ashland, OR, USA). S1A Fig and Fig 1A show representative gating examples.