Igd 11 26c 2a

For analysis of lymphocyte populations, single-cell suspensions were prepared from homogenised spleens or bone marrow. For splenic suspension preparation, erythrocytes were destroyed with lysis buffer (BD Biosciences). The cells were treated with the appropriate combination of the following antibodies: CD16/32 (Fc block) (93) (eBioscience), B220 (RA3-6B2) (eBioscience), CD19 (eBio-1D3) (eBioscience), IgD (11-26c.2a) (eBioscience), IgM (II/41) (BioLegend), CD43 (S7) (BioLegend), CD24 (M1/69) (BD Biosciences), CD21 (7G6) (BD Biosciences), and BP-1/Ly-51 (6C3) (BioLegend). To identify splenic plasma cells or plasma cells from lymph node in vivo, cells positive for CD138 (281.2) (eBioscience) and negative for IgD (11-26c.2a) were considered plasma cells. For analysis of in vitro B-cell cultures, after blocking Fc receptors using anti-CD16/32 antibody, CTV-labelled cells were stained with the antibodies CD138 (281.2) and IgG1 (A85.1). For detection of vimentin using flow cytometry, anti-vimentin antibody (ERP3776) (Abcam) was used.

Bones, spleen, and thymus were dissected, crushed in PBS with 2% FCS and cells were collected after passing through a 70 μm filter. They were then Fc-blocked (CD16/32; 93) and stained with combinations of the antibodies Sca1 (D7), CD105 (MJ7/18), CD41 (MWReg30), CD48 (HM48-1), CD3 (145-2C11), CD4 (RM4-5), CD8 (53-6.7), B220 (RA3-6B2), NK1.1 (PK136), Mac1 (M1/70), Gr1 (RB6-8C5), TER119 (TER-119), CD150 (TCF15-12F12.2), CD117 (2B8, eBioscience), CD127 (A7R34), CD44 (IM7), CD25 (PC61.5, eBioscience), CD19 (1D3, eBioscience), TcRβ (H57-597, eBioscience), TcRγδ (GL3, eBioscience), Ly6C (AL-21), Ly6G (1A8), MHCII (M5/114.15.2), CD11c (N418), PDCA1 (927), Ly6D (49H4), Flt3 (A2F10), IgD (11-26c.2a), and IgM (11/41, eBioscience). All antibodies were purchased from BD Biosciences unless otherwise indicated. Propidium iodide (PI) was utilized to discriminate dead cells. For hematopoietic stem and progenitor cell isolation, cells were subjected to lineage depletion using Dynabeads sheep anti rat IgG (Life Technologies) together with TER119, CD19, CD3, Gr1, and CD11b antibodies prior to staining. Analysis and cell sorting was performed primarily on an LSR Fortessa and FACSAria IIu (BD Biosciences). Analysis of data was done using the Flowjo 9.9.6 software (Flowjo).