Cd11a fitc clone m17 4

Spleens were collected in ISCOVEs media and mashed through mesh screens to obtain single cell suspensions. Cells were incubated with red blood cell (RBC) lysis buffer to lyse red blood cells which was stopped by adding media. The cells were then resuspended in complete ISCOVEs media. The cells were counted using a hemocytometer and an aliquot was taken for staining with extracellular molecules using fluorochromes CD11a-FITC–clone M17/4, (Biolegend, San Diego, CA), CD44-PE–clone PE, CD4-PE-Cy5–clone GK1.5, and CD62L-PE-Cy7–clone MEL-14 (Tonbo Biosciences, San Diego, CA), to determine T cell activation. For intracellular cytokine staining, aliquots for each sample from the counted cells were stimulated in vitro with a cell stimulation cocktail (Tonbo Biosciences) for 5 h. After 5 h of stimulation, cells were stained with CD4-FITC–clone GK1.5 and incubated for 40 min in the fridge before fixation with 2% Paraformaldehyde. Cells were then permeabilized using perm/wash buffer (Tonbo Biosciences, San Diego, CA), and incubated with Fc block for 20 min followed by a 40-min incubation with IFNγ PE–clone XMG1.2, and TNF-PE-Cy7–MP6-XT22, or IL-2-PE–JES6-5H4, and IL-10 FITC–clone JES5-16E3 (all from Biolegend). Data was collected on an FC500 (Beckman Coulter, Indianapolis, IN) flow cytometer and analysed by FlowJo (Ashland, OR).

CD3-FITC clone 145–2C11, CD4-PE-Cy7 clone RM4–5, CD4-PE clone RM4–4, CD11a-FITC clone M17/4, CD49d-PE clone R1–2, CXCR5-biotin clone L138D7, PE-Cy7 streptavidin, CD44-FITC and CD44-APC-Cy7 clone IM7, PD-1 APC clone 29F.1A12, GL7-PacBlue and GL7-PE clone GL7, CD19-PerCP-Cy5.5 clone 6B2, CD138-BV421 clone 281–2, IgD-APC-Cy7 clone 11–26c2a, CD95-PE clone SA367H8, CD73-BV605 clone TY/11.8, CD71-BV421 Clone RI7217, and CD80-PE clone 90, were purchased from Biolegend (San Diego, CA). BcL6-PE-CF594 clone K112–91 was purchased from BD Biosciences, San Jose, CA)