Anti igd bv786

Lymph nodes (axillary or popliteal) were collected in FACS buffer (PBS1X, 10%FCS, 2mM EDTA) on ice. Single-cell suspensions were obtained by mechanical disruption through a 70-mm cell strainer (BD Biosciences). Erythrocytes were lysed with 1 ml of ACK lysing buffer (Gibco). After incubation with 5 μg/ml of anti-CD16/32 (rat mAb 2.4G2, mouse Fc block BD) for 15 min at 4°C, cell surface antigens were stained for 30 min at 4°C. When a biotin antibody was used, the cells were then incubated with a streptavidin-fluorophore conjugate for 20 min at 4°C. Flow cytometric analysis was performed on a BD LSRFortessa. Antibodies used: from BD; Anti-IgM-e710 (R6-60.2, 550881), Anti-IgD-BV786 (11-26c.2a, 563618), Anti- T-and B-cell activation antigen-FTIC (GL7, 553666), Anti-Igλ1,2,3-BV650 (R26-46, 744526), Anti-CD95-PE-Cy7 (Jo2, 557653) streptavidin-BV711 (563262), streptavidin-AF647, from Biolegend; Anti-CD38-PB (90, 102719), Anti-CD138-BV421 (281-2, 142508), Anti-B220-BV605 (RA3-6B2, 103244) live/dead marker Zombie NIR (Biolegend, 423106) and from eBiosciences; Anti-T-and B-cell activation antigen-e660 (GL7, 50-5902-82), Anti-CD4-eF780 (RM4-5, 47-0042-82), Anti-CD8-eF780 (53-6.7, 47-0081-82), Anti-NK1.1-eF780 (PK136, 47-5941-82), Anti-F4/80-eF780 (BM8, 47-4801-82), Anti-TM4-Core-biot (5μg/mL) provided by A.T.M and L.S (Fred Hutchinson Cancer Research Center, Seattle).