Cd21 pe

Peripheral B-cell phenotyping was conducted using flow cytometry. 20 µl of normal serum mix consisting of mouse and goat serum (Dako) were pipetted in each FACS vial with the cell suspension, mixed well, and incubated for 10 minutes at room temperature in the dark. Subsequently, 39.5 µl of an antibody mix made of IgD FITC, CD21 PE, CD5 Per-Cy5.5, CD38 PE-Cy7, IgM APC, CD27 APC-H7, CD19 AmCyan (BD Biosciences, San Jose, California, USA), and CD24 Pacific Blue (EXBIO Praha, Vestec u Prahy, Czech Republic) were pipetted into the vials and mixed well. After another 15 minutes of incubation as described above, lysis buffer (BD Biosciences, San Jose, California USA) was added and the vials were incubated for another 10 minutes. After centrifugation at 250×g for 5 minutes, the supernatant was poured off and cells were suspended in 3 ml of PBS and mixed. After another round of washing the supernatant was discarded and the cell pellet was loosened and fixed by adding 250 µl of PBS with 1% formaldehyde. After fixation, measurement was carried out using a FACS Canto II and analyses were performed with BD Facs Diva.

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).