Anti mtor antibody

For surface marker detection, cells were stained with anti-CD45, anti-CD4, anti-CD8, anti-B220, anti-CD11b, anti-F4/80, anti-Ly-6C, and anti-Ly-6G antibodies. For Th1 and Th17 cell analysis, cells were stimulated with phorbol-12-myristate-13-acetate (50 ng/mL) and ionomycin (500 ng/mL; Sigma) in the presence of brefeldin A (BD Bioscience, San Diego, CA, USA) for 5 h. Then, the cells were incubated with an anti-CD4 antibody, permeabilized with permeabilization buffer (Thermo Fisher Scientific), and incubated with anti-IFN-γ and anti-IL-17A antibodies. Treg cells were detected using a mouse Treg cell staining kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. For determining intracellular levels of mTOR, CD4⁺ T cells were permeabilized with permeabilization buffer and stained with anti-mTOR antibody (Cell Signaling Technology, Danvers, MA, USA) for 60 min, followed by the secondary antibody, a Alexa-Fluor 647-conjugated antibody (Thermo Fisher Scientific) for 30 min in the dark at 4°C. For the glucose uptake assay, prestained cells were incubated with 10 μM 2-NBDG (Thermo Fisher Scientific) for 30 min, followed by flow cytometric detection of fluorescence produced by the cells. All stained cells were analyzed by flow cytometry on a BD FACSCalibur (BD Bioscience).