Cell suspensions of in vitro stimulated splenic B cells were washed in phosphate-buffered saline (PBS). To reduce Fc receptor-mediated binding by antibodies of interest, B cells were preincubated for 15 min with 0.5 μg/ml anti-mouse CD16/CD32 (Clone 2.4G2, BD Pharmingen, ref 553142) in FACS buffer [PBS supplemented with 2% FCS and 2 mM (ethylenedinitrilo)tetraacetic acid (EDTA) (Sigma-Aldrich)]. Cells were then labeled with 0.5 μg/ml anti-mouse B220-BV421 (clone RA3-6B2, BioLegend, ref 103240) and 0.5 μg/ml anti-mouse IgG1-FITC (clone A85-1, BD Pharmingen, ref 553443) antibodies or with 0.25 μg/ml anti-mouse B220-APC-Cy7 (clone RA3-6B2, BD Pharmingen, ref 552094) and 0.5 μg/ml anti-mouse IgG1-FITC (clone A85-1, BD Pharmingen, ref 553443) or 0.5 μg/ml anti-mouse IgG3-FITC (clone R40-82, BD Pharmingen, ref 553403) and anti-mouse IgG2a/2b-BV421 (clone R2-40, BD OptiBuild, ref 744292) antibodies.
After 45 min, cells were washed in PBS and suspended in FACS buffer. For nonviable-cell exclusion, 7-AAD (BD Pharmingen, ref 559925) was added on cells 10 min before flow cytometry analysis. Data were acquired on a BD Pharmingen Fortessa LSR2 (BD Biosciences, San Jose, CA, USA) and analyzed using Flowlogic™ software (Miltenyi Biotec).
After 45 min, cells were washed in PBS and suspended in FACS buffer. For nonviable-cell exclusion, 7-AAD (BD Pharmingen, ref 559925) was added on cells 10 min before flow cytometry analysis. Data were acquired on a BD Pharmingen Fortessa LSR2 (BD Biosciences, San Jose, CA, USA) and analyzed using Flowlogic™ software (Miltenyi Biotec).