Anti cd43

Bone marrow, spleen, mesenteric lymph node (mLN) and Peyer’s Patch (PP) cells were isolated as described previously (26 (link)). In brief, cells were stained with anti-B220 (BioLegend), anti-CD11b (BD Biosciences), anti-CD43 (eBioscience), anti-CD24 (BioLegend), anti-BP1 (eBioscience), anti-IgD (BioLegend), anti-IgM (BioLegend), anti-CD93 (eBioscience) and anti-CD23 (BioLegend) for B cell development staining. For B1 and B2 cell development staining, cells were stained with anti-B220 (BioLegend), anti-CD19 (BioLegend), anti-CD43 (eBioscience), anti-CD23 (BioLegend) and anti-CD5 (BioLegend). For FOB and MZB cell staining, cells were stained with anti-B220 (BioLegend), anti-CD19 (BioLegend), anti-CD21 (BD Biosciences) and anti-CD23 (BioLegend).Cells were stained with anti-B220 (BioLegend), anti-Gl7 (BioLegend), anti-CD95 (Fas) (BD Biosciences), anti-CD86 (BioLegend), and anti-CXCR4(BioLegend) for GC B cell staining and cells were stained with anti-B220 (BioLegend), anti-MHC II (eBioscience) and anti-CD86 (BioLegend) for B cell activation staining.

Wild-type soluble ICAM-1-His6 (D1-D5) was expressed in 293 cells and purified on Ni-NTA agarose^{3 (link)}. Human SDF1-α was from R&D System. Cytochalasin D was from Santa Cruz. Blebbistatin was from AbCam. Phalloidin-Alexa488 was from Invitrogen. Anti-CD43 was from ebiosciences. Glass-bottom dishes and plates were from Mattek. Leibovitz’s L-15 medium and RPMI-1640 medium were from Life Technologies. The reagents for the lentiviral Gateway system were from Life Technologies. Nucleofector Kit V was from Lonza.

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.