Fix perm reagent kit

The immune cell phenotyping and analysis of functional markers were performed by multiparametric flow cytometry analysis. Briefly, cells isolated from the spleen following RBC lysis were incubated at 37°C, 5% CO₂ for 3–4 h with brefeldin A (GolgiPlug). Cells were first blocked using mouse Fc-block (anti-CD16/32) followed by staining for surface markers. Next, cells were washed, fixed, and permeabilized using the Fix-Perm reagent kit (BD Biosciences, Franklin Lakes, NJ, USA) followed by staining for intracellular markers. The following fluorochrome-conjugated anti-mouse antibodies were used: anti-CD11c APC, anti-CD19 BV605, anti-CD44 FITC, anti-CD62L APC-Cy7, anti-granzyme B BV421, anti-IFN-γ BV711, anti-IL4 PE, anti-B220 BUV737, anti-CD3 BV650, anti-CD4 BV786, anti-CD8 BUV396, anti-Foxp3 PE-CF594, and anti-GL7 PerCp-Cy5.5 (BD Biosciences). FACS data acquisition was done on a five-laser Fortessa X-20 flow cytometer (BD Biosciences) and analyzed using FlowJo version 10 (FlowJo LLC, Ashland, OR). Forward and side scatter parameters were used to set singlets and leukocyte gates. Fixable viability stain BV510 included in the surface antibody cocktail was used to gate out dead cells and analyze only viable cells. Overall gating strategy is shown (Supplemental Figure S1).