Fc blocker clone 2.4g2

The following materials were commercially obtained from the sources indicated: Mitomycin C from Sigma–Aldrich (St. Louis, MO, USA); fetal bovine serum (FBS), mouse IFN-γ ELISA Ready Set Go!, mouse IL-2 ELISA Ready Set Go!, and an Alexa 546-conjugated anti-rabbit IgG antibody from Thermo Fisher Scientific (Waltham, MA, USA); Fc blocker (clone 2.4G2), an FITC-conjugated anti-CD11b antibody, an FITC-conjugated anti-Gr-1 antibody, an anti-CD3 antibody, and an anti-CD28 antibody from BD Biosciences (Franklin Lakes, NJ, USA); an anti-HDC antibody (ab37291) from Abcam (Cambridge, UK).

Mice were s.c. immunized with alum-adsorbed NP–OVA (50 µg) and TLR7–alum or TLR7-NP (equivalent gardiquimod dose, 20 µg) in 100 µl PBS at tail base. Inguinal dLNs were excised at day 4, day 7, day 14 and day 22 to prepare single-cell suspensions. For flow cytometry analysis of GC B cells, follicular T cells (TFH) and plasmablasts, cells from the dLNs were stained with Ghost Dye Violet 510 (Tonbo Biosciences). Cells were then washed and blocked with Fc-blocker (clone 2.4G2, BD Bioscience) prior to staining with markers, including CD19 (clone 1D3/CD19, Biolegend), CD38 (clone 90, BD Biosciences), CD95 (clone Jo2, BD Biosciences), CD138 (clone 281-2, BD Biosciences), CD44 (clone IM7, BioLegend), CD3 (clone 17A2, BioLegend), CD4 (clone GK1.5, BioLegend), CXCR5 (clone L138D7, BioLegend) and PD1 (clone 29F.1A12, BioLegend). After staining, cells were washed and fixed with 1.5% PFA. Stained cells were collected using BD FACS Diva v8.01 software associated with a BD LSRII flow cytometer. Data were analysed with FlowJo 10 software. The gating strategy consisted of gating GCBC on live single CD3⁻CD19⁺CD95⁺CD38⁻ cells, TFH on live single CD19⁻CD3⁺CD4⁺CXCR5⁺PD1^hi, and plasmablasts on live single CD138⁺CD44⁺ cells.