Pe cy7 conjugated anti cd19

For human B cell staining, cells were stained with PE-Cy7-conjugated anti-CD19 (BioLegend, #302216), PE-conjugated CD27 (BioLegend, #302808), BV421-conjugated IgD (BioLegend, #348226), BV421-conjugated anti-CD138 (BioLegend, #356516), and APC-conjugated anti-IgG antibodies (BioLegend, #304136). Alternately, cells were permeabilized and stained with primary antibodies against ORAI1, ORAI2, and STIM2 at 37°C for 30 min. Cells were then washed with phosphate-buffered saline (PBS) and incubated with Alexa Fluor^® 488 Goat antirabbit IgG (Thermo Fisher, #A-11008) secondary antibodies at room temperature for 30 min. For mouse cell staining, cells were stained with APC-conjugated B220 (BioLegend, #318326), PE-Cy7-conjugated CD38 (BioLegend, #102718), BV421-conjugated CD138 (BioLegend, #142523), PE-conjugated PD-1 (BioLegend, #135205), APC-conjugated CXCR5 (BioLegend, #145506), PE-conjugated CD95 (BioLegend, #152608), fluorescein isothiocyanate (FITC)-conjugated GL-7 (BioLegend, #144612), APC-Cy7-conjugated-CD4 (BioLegend, #100526), BV510-conjugated-CD3 (BioLegend, #100234), Percp-cy5.5-conjugated-CD45 (BioLegend, #103132), APC-conjugated-CD8 (BioLegend, #100712), PE-Cy7-conjugated-CD19 (BioLegend, #506921) and BV605-conjugated-CD11b (BioLegend, #101257). Samples were analyzed using a BD LSR Fortessa (BD Bioscience).

Flow cytometry was performed at the UCM Cytometry and Fluorescence Microscopy Unit. Cells were washed with PBS/EDTA 2 mM/0.5% BSA and stained for 15 min at room temperature with the fluorescence‐labelled antibodies or corresponding isotype controls. The following anti‐human monoclonal antibodies (mAbs) were used for flow cytometry: fluorescein isothiocyanate (FITC)‐conjugated anti‐CD1c, anti‐HLA‐DR, anti‐CD123; allophycocyanin (APC)‐conjugated anti‐HLA‐DR; phycoerythrin (PE)‐conjugated anti‐CD11c and anti‐CD303; peridinin‐chlorophyll‐protein (PerCP)‐conjugated anti‐CD14 and anti‐CD4 (Myltenyi Biotec). APC‐conjugated anti‐CD3; Alexa Fluor 488‐conjugated anti‐IFN‐γ and anti‐IL‐4 (BD Pharmigen). PE‐conjugated anti‐IL‐10 and anti‐IL17A; PE/Cy7‐conjugated anti‐CD19 and Alexa488‐conjugated anti‐IL‐17A (BioLegend). For each staining, the corresponding isotype controls (IgG2A‐FITC, IgG1‐PE, IgG2A‐PerCP or IgG1‐APC) were also assayed.