Anti igd

WT and WKO mice with or without expressing Lifeact-GFP were immunized intraperitoneally with sheep red blood cells (SRBC) (Innovative Research Cat# ISHRBC10P) twice 7 days apart and euthanized at 7 days following the second immunization. Splenocytes were released from the spleens using frosted glass slides and filtered through 40 μm cell strainer (Thermo Fisher, Cat# 22-363-547). Red blood cells were lysed using ACK lysing buffer (Gibico Cat# 10492-01). GCBs were enriched using a negative selection method based on a published protocol (Cato et al., 2011 (link)). Briefly, splenocytes were incubated with biotinylated anti-CD43 (eBioscience Cat# 13-0431-82), anti-CD11c (eBioscience Cat# 13-0114-81), and anti-IgD (Southern Biotech Cat# 112008) antibodies. After washing, cells were incubated with anti-biotin microbeads (Miltenyi Biotec Cat# 130-090-485) and went through a LS column (Miltenyi Biotec Cat# 130-042-401) according to the manufacture’s recommended protocol. Cells eluted from LS columns were collected as enriched GCBs.

For FACScan analysis and screening of different gene knockout (KO) cells, the following anti‐human antibodies were used: anti‐CD79A‐PE (HM47, BioLegend), anti‐CD79B‐PE (CB3‐1, BioLegend), anti‐IgM‐APC (G20‐127, BD Biosciences), mouse IgG1, κ isotype control‐APC (MOPC‐21, BD Biosciences), anti‐IgD‐PE (IA6‐2, BD Biosciences), mouse IgG2a, κ isotype control‐PE (G155‐178, BD Biosciences), anti‐Igλ‐APC (1‐155‐2, eBioscience), anti‐CD19‐Alexa fluor 647 (HIB19, BioLegend), and anti‐CD81‐APC (1D6, eBioscience). To stimulate cells and to measure calcium responses, anti‐CD79B (CB3‐1, BioLegend), anti‐CD79B (EPR6860, Abcam), and anti‐CD19‐Alexa fluor 647 (HIB19, BioLegend) antibodies were used. To prepare PLA probes and F(ab′)₂ fragments, the following antibodies were used: anti‐human CD79B (CB3‐1, Acris), anti‐human CD19 (HIB19, Biolegend). The following antibodies were used for Western blot (WB): anti‐IgM (Southern Biotech), anti‐IgD (Southern Biotech), anti‐lambda (Southern Biotech), anti‐CD79A (HM47, BioLegend), anti‐CD79B (CB3‐1, Southern Biotech), anti‐BLNK (2B11, Santa Cruz Biotechnology), and anti‐GAPDH (6C5, Thermo Fisher).