Cd23 pe

After 6 days culture, cultured cells were analyzed for expression of surface markers by multi-color flow cytometry. Cell aliquots were treated with anti-human Fc mAb for 20 minutes and stained for 30 minutes with selected combinations of fluorochrome-conjugated antibodies.
For the analysis of B cell subpopulations and activation markers, the monoclonal antibodies used were CD19-PE-CF594 (BD Biosciences, San Jose, CA), CD62L-PE-Cy5 (BD Biosciences), IgD-FITC (BD Biosciences), CD27-PE-Cy7 (BD Biosciences), CD24-PE (BD Biosciences), CD38-PerCP-Cy5.5 (Beckman Coulter, Brea CA), CD138-PE-Cy7 (eBioscience, San Diego, CA), CD23-PE (BD Biosciences), CD21-PE-Cy5 (BD Biosciences), IgG-PC7 (BD Biosciences), CD86-Alexa 700 (BD Biosciences) and CD95-Pacific Blue (BioLegend, San Diego, CA).
For plasma cell staining, cultured cells were first incubated with monoclonal antibodies to surface markers (CD38, CD138 and CD19), fixed and permeabilized with Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences) and then incubated with Blimp-1-PE (R&D systems) and Pax5-APC (eBioscience) for 30 minutes.
For the proliferation assay, 1–10×10⁶ cells of interest were labeled with 1μM carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen) before culture initiation. After 6 days of culture, cells were harvested, incubated with antibodies, and dilution of CFSE was assessed by flow cytometry.

Phenotypic analysis of cell suspensions was performed by flow cytometry. Cells were stained with a panel of seven 8-colour antibody combinations (Table 1). The following 6 monoclonal antibodies were used as a “backbone” in all combinations: CD38-PerCP5.5, CD10-APC, CD19-APC-H7, CD5-V450, CD45-V500 (BD Biosciences) and CD27-PeCy7 (Beckman Coulter). All FITC- or PE-labelled antibodies were specific of a particular combination, i.e. CD20-FITC, CD44-FITC, CD24-PE, CD40-PE, (Beckman Coulter); CD43-FITC, CD81-FITC, CD86-FITC, CD21-PE, CD22-PE, CD23-PE, CD268 (BAFF-R)-PE, IgD-PE, (BD Biosciences) IgM-FITC (Dako). Clone and isotype specificity of these antibodies are detailed in S1 Table; the antibodies were used at the dilution recommended by the manufacturers.
Samples containing 10⁶ cells in a volume of 100 μl were incubated with the combination of antibodies at the supplier recommended concentration during 20 minutes in the dark, at 4°C. After erythrocytes lysis with NH₄Cl at 4°C for 5 minutes, samples were washed with HBSS medium (Gibco).
Analysis was performed using 3-laser, 8-colour BD FACSCanto II flow cytometer (BD Biosciences) and FACSDiva software version 6 (BD Biosciences). At least 10⁴ cells were acquired in the CD19⁺ gate. BD CompBeads (BD Biosciences) were used for compensation settings. Cytometer performances were checked daily using CST beads (BD Biosciences).