Cd24 m1 69

Fluorochrome-or biotin-labeled monoclonal antibodies against the following antigens: CD8 (53-6.7), CD44 (IM7), CD62L (MEL-14), CD124 (mIL4R-M1), CXCR3 (CXCR3-173), and CD24 (M1/69) were purchased from BD Bioscience (San Jose, CA, USA), eBioscience (San Diego, CA, USA), and BioLegend (San Diego, CA, USA). Single-cell suspensions were labeled with antibodies for 30 min at 4℃. For intracellular labeling, prepared cells were resuspended in a mixture of fixation and permeabilization buffers from the Foxp3 staining buffer kit (eBioscience, San Diego, CA, USA). Then, intracellular labeling was performed using antibody Eomes (BD Bioscience, San Diego, CA, USA). Flow cytometry was performed on a FACSCalibur and LSR Fortessa (Becton Bioscience, Mountain View, CA, USA). The data were analyzed using the FlowJo software (TreeStar, Ashland, OR, USA).
For the intracellular cytokine assay, CD8 T cells isolated from mouse spleens were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) and 1.5 µM ionomycin (Sigma-Aldrich, St Louis, MO, USA) for 4 h at 37℃ in a CO2 incubator, followed by an additional incubation in the presence of 10 µg/mL brefeldin A (Sigma-Aldrich, St Louis, MO, USA) for 2 h. Cultured cells were labeled with anti-CD8 and anti-CXCR3, followed by fixation, permeabilization, and intracellular cytokine labeling with anti-IFN-γ.