Cd21 apc 7g6

Spleen and lymph nodes (LN) were disrupted and filtered from cell debris through a 70 μm cell strainer to prepare cell suspensions. BM cells were isolated from hind limbs. Bones were flushed with PBS + 2% FCS and filtered through a 70 μm cell strainer. Peripheral blood (PB) was obtained from the retro orbital venous plexus. Erythrocytes were removed using a lysis buffer (BD). B cells were purified by negative selection using EasySep™ Mouse B Cell Isolation Kit (Stemcell). Fo and MZ B cell subpopulations were purified with FACSAria (BD) using CD23-PE (B3B4), CD21-APC (7G6), B220/CD45R-APC-Cy7 (RA3-6B2), CD19-PerCP-Cy5.5 (1D3), all BD. After immunization with NP-CGG, GC B cells (B220^high, IgD^-, CD38^low/-, PNA⁺, CD95⁺) were sorted by FACSAria (BD) using B220/CD45R-APC-Cy7 (RA3-6B2), IgD-V450, CD38-PE, peanut agglutinin (PNA-FITC) and CD95-PE-Cy7 (Jo2).

Cells were incubated with Fc block (anti-CD16/32, BD) and stained with fluorochrome-conjugated monoclonal antibodies in PBS/2% FBS using the following antibodies: CD5 Brilliant Violet 421 (S3-7.3), CD19 PE (1D3), CD19 FITC (1D3), CD23 Brilliant Violet 421 (B3B4), CD21 APC (7G6), CD43 APC (S7) (all BD), B220 APC-eFluor 780 (RA3-6B2), CD93 APC (AA4.1) (all eBioscience), and IgM FITC (polyclonal) (Southern Biotech). Data were analyzed in FlowJo v9.6.4 (Treestar).