Anti cd4 apc efluor 780

Single cell suspensions of total splenocytes, enriched B cells or BM were stained with different combinations of the following antibodies: Anti-CD4 APC-eFluor 780, anti-CD8 APC-eFluor 780, anti-Gr1 APC-eFluor 780, anti-F4/80 APC-eFluor 780, anti-B220 APC, anti-B220 APC-eFlour 780, anti-B220 FITC, anti CD19 PeCy7, anti-CD38 Alexa Fluor 700, anti-CD93 APC, anti-IgM PerCP-eFluor 710, anti CD21/CD35 eFluor 450 (eBiosciences), anti-CD23 PE (BioLegend), anti-CD4 PE-CF594, anti-CD8 PE-CF594, anti-Ly-6G and Ly-6C PE-CF594 and anti-IgG1 BV421 (BD biosciences). Live dead aqua stain was added to separate dead cells (Life Technologies) and eOD-GT8-specific cells were visualized by the addition of FITC-conjugated eOD-GT8 and PE-conjugated eOD-GT8 CD4bs knock-out. BG505 SOSIP- (Sok et al., 2014 (link)) and 2cc-specific memory B cells were visualized by the addition of biotinylated protein with the addition of streptavidin conjugated PE and APC respectively (BD Biosicences).

GBM tumor samples were collected and kept in cold PBS before processing the samples for FACS analysis. Tumor samples were gently dissociated using 70 μm cell strainers (Fisher Scientific 352350) in cold FACS buffer (PBS + 2 mM EDTA + BSA 2%) and washed two times in FACS buffer (50 mL). Cells were incubated for 5 minutes with human Fc block (BD, 564219) on ice and then incubated with antibodies at 4°C for 30 mins. Cells were washed, and 7AAD (BD, 559925) was added (1:100 dilution) before FACS acquisition.
Blood samples were collected on heparin tubes and mix at a 1:1 ratio with DMEM medium at room temperature to perform lymphocyte (BD, NC9587917) PBMC isolation. PBMC were washed in FACS buffer and stained similarly to the tumor samples. Antibodies used were APC anti-CD45 (Biolegend, dilution 1:200), PE-Cy7 anti-CD8a (eBiosciences, dilution 1:200), APC-eFluor 780 anti-CD4 (eBiosciences, dilution 1:200). Data were acquired on BD Fortessa LSRII and analyzed by first gating live cells, then CD45⁺ cells, and finally CD4⁺ cells in the X axis and CD8⁺ in the Y axis of the scatter plot. Analyses were conducted using FlowJo v. 10.6.2.

CD4⁺ cells were depleted using anti-CD4 monoclonal depletion antibody (clone: GK1.5) or matching isotype control (Rat IgG2B). IL-17A neutralization was performed using anti-IL-17A monoclonal antibody (clone: 17F3) or matching isotype control (Mouse IgG1). Antibodies were administered intraperitoneally, 500μg per day every 3 days for a total of 3 doses. Efficacy of CD4⁺ depletion was determined in colonic lamina propria and spleen by flow cytometry. For intestinal lamina propria lymphocytes isolation, tissue pieces were washed with cold PBS and incubated in RPMI with 1 mg/ml Collagenase/Dispase for 30 min at 37°C with shaking at 200 rpm. Splenocytes were disrupted into single cell suspension by passing the organ through 70 μm filter and RBCs were lysed in ACK lysis buffer (Invitrogen) for 3 min. Cells were stained using the following monoclonal fluorescence-conjugated antibodies: BUV395 anti-CD45.2 (Clone: 104, BD Biosciences), APC-eFluor 780 anti-CD4 (Clone: RM4–5, eBioscience), and APC anti-CD8a (Clone: 53–6.7, eBioscience). All antibodies were diluted in FACS buffer (2% FBS, 0.01 Sodium Azide, PBS). Dead cells were gated out by using the Fixable Violet Dead Cell Stain Kit (Invitrogen). Samples were acquired on the BD LSRFortessa (BD Biosciences) and analyzed with FlowJo Software (Treestar).