Profiling Lymphoid Cell Subsets

Single cell suspensions were obtained from lymphoid organs by gently passing through a 40 μM cell strainer in PBS containing 0.5% BSA and 0.5 mM EDTA. Cells were incubated for 10 min with Fc receptor‐blocking antibody (TruStain fcX; Biolegend, San Diego, CA) and stained with the following fluorescently labeled antibodies: anti‐mouse B220 (RA3‐6B2), CD3 (17A2), CD45.1 (A20), CD19 (6D5), CD21/CD35 (7E9), CD23 (B3B4), IgM (RMM‐1), IgD (11‐26c.2a), CD5 (53‐7.3), all from Biolegend, and CXCR4 (2B11) from eBioscience (San Diego, CA). Alexafluor 647 Mouse IgG2b, k (MPC‐11; Biolegend) was used as isotype control. For CXCR4 intracellular staining, cells were fixed with PFA 4% in PBS for 15 min at 4°C and permeabilized with saponin 0.2% in PBS containing 0.5 mM EDTA and 0.5% BSA. Acquisition was performed on a MACSQuant Analyzer (Milteny Biotec) and data analysis was done using FlowJo vX.0.7 software (Treestar, Ashland, OR). All flow cytometry experiments were performed adhering to the “Guidelines for the use of flow cytometry and cell sorting in immunological studies” [47 (link)].