Anti mouse cd16 cd32 monoclonal antibody

Omental tumour and LN tissues were mashed through a 70 µm cell strainer to acquire single cell suspension. Cells were centrifuged at 500 rcf for 5 min at 4 °C, washed with FACS buffer twice, then resuspended in 85 µL FACS buffer. Cells were then incubated with anti-mouse CD16/CD32 monoclonal antibody (1:200, BD Biosciences, Cat# 553142) for 15 min at 4 °C. Antibodies or respective isotype controls listed in Supplementary Table 1 were diluted in FACS buffer and used to surface stain cells for 20 min at 4 °C. Precision Count Beads (Biolegend) were additionally added to allow quantification of the total number of immune cells in each sample. BD Fortessa X-20 flow cytometer and FlowJo software were used to analyse samples. Immune cell populations were defined as listed in Supplementary Table 2.

In order to avoid variations linked to sex (20 (link), 21 (link)), female BALB/c mice aged 6-8 week were vaccinated at day 0 via i.m. route with MO (5μg), Omp19 (5μg), Omp19+Al+CpG (5µg+50µg+25µg), and PBS (MO, Omp19, Omp19+Al+CpG, n=5; PBS, n=3). Draining lymph nodes (DLNs) on day 4 were harvested to prepare single-cell suspensions. The single-cell suspensions were blocked with anti-mouse CD16/CD32 monoclonal antibody (BD Biosciences, Franklin Lakes, USA). The live/dead fixable near-IR dead cell stain kit (Invitrogen, Carlsbad, USA) was used to exclude dead cells from the data analysis. For GC B cell staining, cells were incubated with Brilliant Violet 421™ anti-mouse CD4 (Biolegend, clone GK1.5, San Diego, USA), FITC anti-mouse CD95(Fas) (Biolegend, clone SA367H8, San Diego, USA) and APC anti-mouse/human GL7 (Biolegend, clone GL7, San Diego, USA) for 30 minutes at 4°C. For Tfh cell staining, cells were incubated with PE/Cyanine7 anti-mouse/human CD45R/B220 (Biolegend, clone RA3-6B2, San Diego, USA), PerCP/cy5.5 anti-mouse CD185 (CXCR5) (Biolegend, clone L138D7, San Diego, USA) and PE anti-mouse CD279 (PD-1) (Biolegend, clone RMP1-14, San Diego, USA) for 30 min. After washing, cells were acquired on a BD canto II (BD Biosciences, USA), and data were analyzed in FACS Diva™ Software.