Cell quest pro software

The apoptosis of microglia was evaluated using an Annexin V-FITC/PI apoptosis detection kit (BD Biosciences, CA, USA) via flow cytometry analysis [38 (link)]. After washed with phosphate buffered saline, microglial cells with indicate treatments were resuspended in binding buffer and then stained with Annexin V-FITC (5 µL; 10 min) and propidium iodide (PI) (5 µL; 5 min) at room temperature in the darkness according to the manufacturer’s instructions. The apoptosis rate of stained microglia was analyzed by a flow cytometry (FACScan, BD Biosciences) using Cell Quest Pro software (Beckman Coulter, CA, USA). All experiments were repeated three times.

Cells were aliquoted into tubes and washed once in phosphate buffered saline (PBS). For Th17 analysis, the cells were incubated with fluorescein isothiocyanate (FITC) anti-human CD4 (eBioscience, San Diego, CA, USA) at 4°C for 20 min. For Treg analysis, the cells were incubated with PerCP anti-human CD4 (eBioscience, USA) and FITC anti-human CD25 (eBioscience, San Diego, CA, USA) at 4°C for 20 min. After the surface staining, the cells were then fixed and permeabilized with Perm/Fix solution (Beckman Coulter) and were stained with PE anti-human IL-17A (eBioscience) for Th17 detection or PE anti-human Foxp3 (eBioscience, CA, USA) for Treg detection. All staining was performed according to manufacturer’s protocols. Isotype controls were used to enable correct compensation and confirm antibody specificity. Samples were analyzed using a FACS Calibur flow cytometer and Cell Quest Pro software (Beckman Coulter).