Anti foxp3 apc or pe

For the detection of CD8+ T cell expansion in the blood, lymphocytes were washed and incubated with 10 mg/mL 2.4G2 FcR block for 15 min at 4 C. Fluorochrome-labeled antibodies to surface markers were then added for 20-30 min in PBS + 1% BSA and the cells washed once with PBS/1% BSA. Red blood cells were lysed with either ammonium chloride buffer (in house) or with Erythrolyse Red Blood cell lysis buffer (AbD SeroTec) and samples washed once with PBS/1% BSA. Samples were then run on a BD FACSCanto II or FACSCalibur and data analyzed using FCS Express or FACs Diva software. Staining of lymphocytes from spleens and tumors were performed similarly. In some cases, tumor samples were additionally stained with a fixable live/dead stain (eBioscience) to visualize only the live cell population. For intracellular staining, cells were surface stained and then fixed and intracellularly stained using the anti-Mouse/Rat Foxp3 Staining Set (BD Biosciences). Antibodies were anti-CD4 eF450 (GK1.5), anti-CD8-APC-eF780 or -FITC (53-6.7), anti-Foxp3 APC or -PE (FJK-16), anti-4-1BB-PE or -eF450 (17-B5), anti-KLRG-1-APC (2F1), anti-MHCII-APC-eF780 (M5/114.15.2), anti-CD11c-APC (N418), streptavidin-PE-Cy7 (all eBioscience), anti-Ki67 APC (B56) (BD Biosciences), anti-4-1BBbiotin (Biolegend) or isotype controls.