F4 80 bv785

Cells from each tissue were resuspended in PBE 1x (PBS supplemented with 0.5% BSA + 1 mM EDTA) and incubated for 30 min on ice with fluorescently labeled antibodies: CD45-AF700 (BioLegend Cat. No. 103127), CD4-BUV495 (BD Biosciences Cat. No. 565974), CD8α-BUV805 (BD Biosciences Cat. No. 612898), TCRβ-BUV395 (BD Biosciences Cat. No. 742485), NK1.1-BV785 (BioLegend Cat. No. 108749), CD11b-BV711 (BioLegend Cat. No. 101241), CD11c-BUV496 (BD Biosciences Cat. No. 750483), I-A/I-E-BUV395 (BD Biosciences Cat. No. 743876), Ly6G-BV605 (BioLegend Cat. No. 127639), F4/80-BV785 (BioLegend Cat. No. 123141), IFN-δ-PerCP-Cy5.5 (BD Biosciences Cat. No. 560660), TNF-α-PE-Cy7 (BioLegend Cat. No. 506323), IL-17α-BV421 (BioLegend Cat. No. 506925), B220-BV785 (BioLegend Cat. No. 103245), FAS-PE-Cy7 (BioLegend Cat. No. 152617), CD38- PerCP-Cy5.5 (BioLegend Cat. No. 102721) and CD138-BV650 (BioLegend Cat. No. 142517). WA-1 RBD and Omicron RBD biotinylated were purchased from Sino Biological and conjugated with streptavidin. LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, L-34965, was purchased from Life Technologies. Cells were filtered and washed with PBE 1x again before analysis on BD FACS Symphony cytometer. Analyses were performed using FlowJo v. 10 software.

Tumors were harvested and resuspended into single-cell solution in PBS as described above. Murine spleens were mashed through a 70 μm filter into a ACK lysing buffer (Gibco, #A1049201) and incubated for 3 minutes. A total of 10 mL of PBS was added after the RBC lysis and the suspension was centrifuged at 1,500 rpm for 5 minutes. The supernatant was discarded and the pellet was resuspended in PBS. After Fc blocking (BioLegend, 101319; 1:100) and Live/Dead staining with zombie NIR (BioLegend, #423105; 1:1,000) was performed during a 10-minute incubation at 4°C, the samples were washed with PBS. Conjugated antibodies including CD45-BV711 (BioLegend, #103147; 1:200), CD4-PE (BioLegend, #100407; 1:100), CD8-Pe-Cy7 (BioLegend, #100721; 1:100), NK1.1-FITC (BioLegend, #108705; 1:100), B220-Pe-Cy5.5 (BioLegend, #103209; 1:100), CD11b-APC (BioLegend, #101211; 1:200), F4/80-BV785 (BioLegend, #123141; 1:200), GR1-Pe Tx red (BD Biosciences, # 562710; 1:200) were incubated with each sample at 4°C in the dark for 20 minutes and washed with FACS buffer (2% FBS in PBS). The samples were analyzed using BD LSRFortessa X-20 flow cytometer. Collected flow cytometry data were analyzed using FlowJo software.

For detection of antigen-specific GC B cells, freshly isolated LN cell suspensions from individual mice were stained with Aqua Viability Dye (Thermo) and Fc blocked with an anti-CD16/32 antibody (clone 93; BioLegend). Cells were then surface stained with the relevant probes (APC or PE labelled stem, HEL, OVA or gp120 proteins for baiting antigen-specific GC B cells) [10 (link)] and the following antibodies: CD45 APC-Cy7 (30-F11; BD), CD3 BV785 (145-2C11; BioLegend), F4/80 BV785 (BM8; BioLegend), Streptavidin BV785 (BD), B220 BUV737 (RA3-6B2; BD), IgD BUV395 (11-26c.2a; BD), CD38 PE-Cy7 (90; BioLegend), GL7 AF488 (GL7; BioLegend). For detection of Tfh cells ex vivo, freshly isolated LN cell suspensions from individual mice were stained with the following panel: Live/dead Red (Thermo), B220 BV605 (RA3-6B2; BD Biosciences), CD3 BV510 (145-2C11; BioLegend), CD4 BUV737 (RM4-5; BD Biosciences), CXCR5 BV421 (L138D7; BioLegend), PD-1 BV786 (29F.1A12; BioLegend). For Ag-specific Tfh identification [23 (link)], cells were cultured as described above and then stained with the following panel: Live/dead Red (Thermo), B220 BV605 (RA3-6B2; BD), CD3 BV510 (145-2C11; BioLegend), CD4 BUV737 (RM4-5; BD), CD25 BB515 (PC61; BD), OX40 PeCy7 (OX-86; BioLegend). All samples were acquired on a BD LSR Fortessa using BD FACS Diva and data was analyzed in FlowJo v10.