Apc cy7 conjugated anti mouse cd19

Antibodies used for immunohistological techniques were fluorescein-conjugated anti-mouse C3 (Cappel Pharmaceuticals, Aurora, OH, USA), rat anti-mouse EGF-like module-containing mucin-like hormone receptor-like 1 (EMR1, F4/80, MCA497GA, AbD Serotec, Oxford, England, UK), rat anti-mouse CD11b (M1/70, BD Biosciences, San Jose, CA, USA), rabbit anti-mouse proliferating cell nuclear antigen (PCNA, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit anti-Ki67 (RM-9106-s, Thermo Fisher Scientific, Waltham, MA, USA).
Antibodies used for flow cytometry were PE-Cy7-conjugated anti-mouse CD11c, APC-Cy7-conjugated anti-mouse CD19, PerCP5.5-conjugated anti-mouse Ly6C/G (Gr1), BD Horizon V500-conjugated anti-mouse CD11b (BD), phycoerythrin-conjugated anti-mouse CCR2 (R&D Systems Minneapolis, MN, USA), and Alexa fluor 647-conjugated anti-F4/80 (AbD Serotec).

Spleen and lymph nodes were harvested from immunized mice five days after the final boost. B cells were enriched using theEasySep mouse pan-B cell isolation kit (Stemcell Technologies, Vancouver, CA). Cells were then stained with a fluorophore-labeled antibody panel, which includes PerCP-Cy5.5 conjugated anti-mouse IgM (BD Biosciences), PerCP-Cy5.5 conjugated anti-mouse IgD (BD Biosciences), APC-Cy7 conjugated anti-mouse CD19 (BD Biosciences), and a cocktail of phycoerythrin (PE) conjugated antibodies used during sorting as a dump channel: anti-mouse Ly6g (Biolegend, San Diego, CA, USA), PE anti-mouse CD3 (Biolegend), and PE anti-mouse F4/80 (Biolegend) and Alexa647 and Alexa488 ovalbumin (InvivoGen, San Diego, CA, USA). Single dump channel-/CD19+/IgM-/ovalbumin^duel+-stained B cells were FACS sorted and barcoded using the Chromium controller (10× Genomics, Pleasanton, CA, USA) and a sequencing library was prepared according to the manufacturer’s instructions using the Chromium Next GEM single-cell 5′GEM, BCR amplification and library construction kit (10× Genomics, Pleasanton, CA, USA).