Flow cytometry and phosphorylation flow
Following incubation with anti-CD16/CD32 mAb (101319; BioLegend) to block the Fc receptors, splenocytes or BM cells were seeded into 96-well plates at a concentration of 2 × 106/well. The following antibodies (Abs) were acquired from BioLegend: FITC channel: anti-CD127 (135008), anti-CD19 (101506), anti-B220 (103206), anti-Annexin V (640906), anti-CD5 (100622), anti-CD11b (101226), and anti-CD138 (142521). APC channel: anti-CD43 (143208), anti-CD21 (123412). PE channel: anti–BP-1 (108307), and anti-CD23 (101608). PE/Cy7 channel: anti-CD24 (101822). PerCP/Cy5.5 channel: anti-IgD (405710). Brilliant Violet (BV) 510 channel: anti-B220 (103247) and anti-CD45.2 (109838). BV421 channel: anti-IgM (406518). Alexa Fluor 647 (AF647) channel: anti-GL7 (144606). APC/Cy7 channel: anti-CD45.1 (110716). Lastly, the PE-anti-NP (N-5070-1) was acquired from Biosearch Technologies (Novato, CA, USA).
Phosphorylation flow (phosflow) was performed to determine the level of phosphorylated WASP and F-actin in primary B cells. Cells were incubated with anti-B220 to mark B cells and soluble antigen (sAg), which consist of biotin-conjugated F(ab’)2 anti-mouse Ig (M + G) (115-066-068; Jackson ImmunoResearch, West Grove, PA, USA) and streptavidin (16000114, Jackson ImmunoResearch), then activated at 37 °C for 5, 10, and 30 min. Following activation, cells were fixed, permeabilized, and then stained with anti-pWASP (A300-205A; Bethyl Laboratories, Montgomery, TX, USA) and AF488–phalloidin (R37110; Thermo Fisher), followed by goat-anti-rabbit (G/R) IgG AF405 antibody (A-31556, Thermo Fisher).
For the T-bet expression assay, splenic B cells were incubated with 10 μg/ml F(ab’)2 goat anti-mouse (G/M) IgM (115-156-020, Jackson ImmunoResearch) for 1 and 2 h, respectively. Subsequently, the splenic B cells were fixed, permeabilized, and stained with anti-B220 as well as anti-T-bet (644809, BioLegend). Samples were collected using a Attune™ NxT sonic focused flow cytometer (Thermo Fisher) and data were analyzed using the FlowJo 10 software (BD Biosciences, Franklin lakes, NJ, USA).