Anti cd8a fitc mab

The spleen was removed from freshly euthanized mice and smashed in 10 mL RPMI-1640 medium supplemented with 10% FBS using Stomacher® 80 Biomaster (Seward Laboratory Systems Inc., Port St. Lucie, FL). To obtain single cell suspensions, tissues were passed through a 70-μm cell strainer. After red blood cell (RBC) lysis, cells were stained with anti-CD19 mAb, anti-CD3 PE mAb, anti-CD4 FITC mAb, anti-CD8a FITC mAb, anti-FOXP3 mAb, anti-ly6C FITC mAb, anti-CD11b PE mAb, anti-ly6G FITC mAb, and/or anti F4/80 FITC mAb (eBioscience, San Diego, CA) on ice for 30 min. For mitochondrial ROS measurement, BMDM were cultured in 6-well plate. On day 7, cells were stimulated with H₂O₂ (250 μM) for 4 h. After staining with MitoSOX Red Mitochondrial Superoxide indicator (5 μM) (Life technologies, Carlsbad, CA), cells were incubated at 37 °C for 20 min and washed with PBS twice. Cells were detached by adding 1 ml accutase (Innovative Cell Technologies, Inc) for 30 min, collected by centrifugation, and resuspended in 0.2 ml FACS buffer. Stained cells were subjected to flow cytometry analysis using a Cytomics FC 500 flow cytometer and CXP software version 2.2 (Beckman coulter, Brea, CA). Data were collected for 10,000 live events per sample.