Anti cd95 pecy7

In brief, spleens were prepared for flow cytometry by generating single cell suspensions. RBCs were lysed by Tris Ammonium Chloride. Single cell suspensions of bone marrow cells were prepared by flushing the marrow followed by RBC lysis. Resulting single cell suspensions of splenocytes or bone marrow cells were Fc blocked with Trustain FcX (anti-mouse CD16/32 clone 93- Biolegend) and stained with combinations of the following antibodies: anti-B220-PacBlue (RA3–6B2), GL7-FITC, anti-CD95-PeCy7 (Jo2), anti-CD4-Alexa Flour 700 (RM4–5), anti-CXCR5-biotin (2G8), anti-PD-1-PE (29F.1A12), anti-CD11c-BV421 (N418), anti-NK1.1-biotin (PK136), anti-Ly6G-BV711 (1A8), CD86-PeCy5 (GL-1), anti-CD44-BV605/APC (IM7), anti-CD62L-PeCy7 (MEL-14), anti-CD80-PE (16–10A1), anti-CD19-BV605 (6D5), anti-CD267-PE (8F10), anti-CD138-PeCy7 (281–2), and SA-PeCy5 from Biolegend; anti-CD43-FITC (S7), anti-Ly6C-FITC (AL-21), anti-CD11b-AF700 (M1/70), anti-CD90.2-biotin (53–2.1), anti-CD19-biotin (1D3), anti-CD40-FITC (HM40–3), anti-IgD-APC (11–26c.28), and SA-V500 from BD Biosciences. Viability staining was performed by incubating samples with Fixable Viability Dye (e780) from eBioscience. Data were acquired on an LSRII flow cytometer (BD Biosciences) and data were analyzed using FlowJo software (Tree Star).

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).