Multiparametric Flow Cytometry Analysis

Surface receptors on live cells were stained with fluorochrome-conjugated antibodies described in Table S2. Parallel detection of mitochondrial mass and potential was carried out by incubating surface-stained cells with fluorescent molecular probes (see below). Intracellular antigens, such as mTOR substrates pS6RP and pAkt, and transcription factors FoxP3 and Helios, were detected following permeabilization and fixation using FixPerm kit (eBioscience Cat Nos 00-5123-43, 005223-56, 008333-56). In agreement with a recent study^{123 (link)}, we have noted far greater sensitivity and accuracy and less variability of flow cytometry as compared to western blot detection of mTORC1 and mTORC2 activities^{13 (link),15 (link),100 (link)–102 (link)}. Intracellular cytokine production was assessed following 3 h stimulation with phorbol 12-myristate 13-acetate (PMA, 5 ng/ml; Sigma, cat. no. P-8139) and ionomycin (500 ng/ml; Sigma, cat. no. I-0634) in the presence of brefeldin A (10 ng/ml, Sigma cat no. B6542). Subsequently, cells were i) stained with antibodies directed to surface antigens; ii) fixed in 1% paraformaldehyde; iii) permeabilized with the eBioscience FixPerm kit; and iv) stained with antibodies directed to cytokines (Table S2).