Multiparametric Flow Cytometry Analysis of Immune Cell Subsets

Surface and intracellular staining were performed as previously described [28 (link)]. Single immune cell populations from the spleen, lymph nodes or tumors were separated with a BD FACSAria II Cell Sorter. Flow cytometric analyses were performed with Flowjo (Tree Star). The following antibodies were used for cell staining: anti-CD3 (clone 145-2C11), anti-CD4 (clone RM4-5), anti-CD8 (clone 53-6.7), anti-CD49b (clone DX5), anti-CD11b(clone M1/70), anti-CD11c (clone HL3), anti-CD19 (clone 1D3), anti-CD25 (clone PC61), anti-CD69 (clone H1.2F3), anti-CD62L (MEL-14), anti-CD44 (clone IM7), anti-Foxp3 (clone FJK-16s), anti-Granzyme B (clone GB11), anti-CCR4 (clone 2G12), anti-CCR5 (clone HM-CCR5), anti-CXCR3 (clone CXCR3-173), NK1.1(clone PK136), anti-F4/80 (clone BM8), anti-Gr-1 (clone RB6-8C5), anti-interferon-γ (IFN-γ, clone XMG1.2), and anti-NK1.1 (clone PK136), CD4 blocking mAb (clone GK1.5), and CD8 blocking mAb (clone 53-6.7).
For detection of phosphorylated S6 proteins, cells from LNs cultured with PMA (10ng/ml) and Ionomycin (500ng/ml) at designated times were immediately fixed with phosflow Lyse/Fix buffer (BD Biosciences) and permeabilized by Phosflow Perm buffer (BD Biosciences). Cells were stained with the Alex488 conjugated antibody for S6P (Ser235,236) (D57.2. 2E; Cell Signaling Technology)