Cd95 pecy7

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).

Flow cytometry on primary murine B cells were performed as described previously [17] (link). Briefly, cells were resuspended in about 100 uL of FACS buffer (PBS with 2% FCS+1 mM EDTA). Fc block was done prior to staining with fluorophores to block FcγIII/II receptors by adding Rat monoclonal anti CD16/32 (eBioscience) for 10 minutes. The cells were washed once with FACS buffer prior to staining with an antibody cocktail. Antibodies used for surface staining of primary B cells include Thy1.1-APC (eBioscience) and CD19-FITC. For staining splenocytes from infected mice, a similar protocol was used. Antibodies in the cocktail for infected cells include GL7-Ax660/APC (eBioscience), CD95-PECy7, CD138-PE, CD3, CD4, CD8-PerCP (BD Pharmingen), B220-Pacific Blue (Southern Biotech) described in [29] (link). CD3, CD4 and CD8 were used in the same fluorophore to eliminate T-cells. This mix is referred to in the text as dump gate.

The antibodies used for flow cytometry included: CD4-PE (RM4-5), CD4-v450 (RM4-5), CD5-PE (53-7.3), CD8-APC (53-6.7), CD19-FITC (ID3), CD22-PE (Cy34.1), CD45R/B220-APC (RA3-6B2), CD62L-APC (MEL-14), CD69-FITC(H1.2F3), CD86-PE (GL1), CD93(AA4.1), CD95-PE.Cy7 (Jo2), CD134-Biotin (OX-86), CXCR5-PE.Cy7 (2G8), PD-1-APC (J43), and PNA-FITC (L7281) (all from BD Biosciences, San Jose, CA), IgM-FITC) (R6-60.2 from Southern Biotech), IgD-APC-Cy7 (11-26c.2a from BioLegend, San Diego, CA, USA), CD21/CD35-eFlour450 [eBio4F3 (4E3)], and CD23-PE-Cy7 (B3B4) (both from eBioscience Inc., San Diego, CA, USA). Biotinylated antibodies were detected using FITC-conjugated streptavidin (BD Biosciences). Flow cytometric analysis was performed using various combinations of these antibodies on single cell suspensions of splenocytes. Stained cells were analyzed in the UNMC Flow Cytometry Research Facility using the BD LSR II flow cytometer. Data were analyzed using FACSDiva software, version 8.0.2 (BD Biosciences). For flow cytometry analyses, splenocytes were collected from mice that were 5–6 months of age.

Cryopreserved spleen suspensions were defrosted and washed twice in buffer, PBS-2% fetal calf serum and incubated with 2.0 μg Fc block per 10⁶ cells for 15 min. The samples were then incubated with the antibody cocktails (1 in 200 dilution per antibody) for 15 min after which they were washed. The majority of samples were processed using the Attune NxT Acoustic Focusing Cytometer, Life Technologies, and the remainder were processed on a BD LSRII. All analyses were performed using FlowJo v10.2.
Antibodies used for the general staining panel: Cd3-AF700 Biolegend 197780, Cd4-PerCP-Cy5.5 Biolegend 100434, Cd5-PE Biolegend 100608, Cd8a FITC Biolegend 100706, Cd19-BV421 Biolegend 115538. Additional antibodies: B220/CD45R-BV510 Biolegend 103247, IgD-APC-Cy7 Biolegend 405716, IgM PE Biolegend 406508, IgG1-FITC BD Bioscience 553443, IgG2a/2b FITC BD Bioscience 553399, IgG3-FITC BD Bioscience 553403, Kappa-AF700 Biolegend 409508, Lambda-APC Biolegend 407306, CD95-PECy7 BD Bioscience 557653, PNA-FITC Vector Laboratories FL-1071. Fc block TruStain fcX™ anti-mouse CD16/32, Biolegend 101320.

Single immune cell suspensions were prepared from spleen or peritoneal lavage after red blood cell lysis. Cells were counted on a BD Fortessa Flow cytometer using ‘Fluoresbrite ^TM Calibration Grade 6.0 micron YG microspheres (Polysciences). Cells were incubated with anti-CD16/CD32 (Fc block, clone 2.4G2; BD Bioscience) plus 2% normal mouse and normal rat serum and stained with various combinations of the following Abs in FACS buffer (PBS containing 2% FBS and 2 mM EDTA) for 30 min at 4°C. Murine reactive antibodies, including B220-Pacific Blue, CD3-PerCPCy5.5, CD4-Pacific Blue, CD5-PerCPCy5.5, CD8-APC Cy, CD11b APC Cy7, CD21 FITC, CD23 PE, CD38-FITC, CD95 PE Cy7, CD138-PE, IgD APC Cy7, IgM FITC, GL-7 biotin, Streptavidin BV500, Ki67-APC, Ly6G PE Cy7, and Ly6C PerCP Cy5.5 were all purchased from BD Bioscience.