Sytox green

To determine whether pyroptosis was involved in the neuroprotection of IPC, the double staining of Annexin V-mCherry and SYTOX green (Beyotime, Shanghai, China) was assessed. After 6 or 24 h following IPC and/or OGD, the BV-2 cells were harvested and seeded, at a density of 5 × 10⁴ cells per well, onto 24-well plates. After being centrifuged at 1000 rpm for 5 min, the cell culture medium was removed and the cells were rinsed with PBS. Then, Annexin V-mCherry binding buffer (194 μL) together with Annexin V-mCherry (5 μL) and SYTOX green (1 μL) was added. After incubation for 20 min at 20 °C in dark, the cells were washed and imaged to determine their fluorescence levels with a confocal microscope (Leica, Wetzlar, Germany). Positive cells were identified by colocalization with the Annexin V–mCherry Red and SYTOX green.

High glucose Dulbecco’s Modified Eagle Medium (DMEM), trypsin and penicillin–streptomycin, were obtained from Macgene (Beijing, China). Fetal bovine serum (FBS) was from PAN-Biotech (Aidenbach, Germany). Complete/Incomplete Freund’s adjuvant and LPS were from Sigma-Aldrich (St Louis, MO, USA). Bovine type II collagen was from Macklin (Shanghai, China). Methotrexate was obtained from Bidepharm (Shanghai, China). 4% paraformaldehyde was from Bioroyee Biotechnology (Beijing, China). EDTA and DAPI were from Solarbio (Beijing, China). Goat anti-rabbit secondary antibody, endogenous peroxidases blocker and goat serum albumin were from ZSGB-Biotech ((Beijing, China). PMA are purchased from Med Chem Express (MCE). Hoechst 33342 was obtained from TargetMol (Shanghai, China). Sytox Green was from Beyotime (Shanghai, China). Primary antibody of IL-6 was from Bioss (Beijing, China). Primary antibody of TNF-α was from Proteintech (Chicago, IL, USA). Primary antibodies against PI3K/p-PI3K were from Bioworld (St. Louis Park, MN, USA). Primary antibodies against p-Akt and p-mTOR were bought from Cell Signaling Technology (Beverly, MA, USA). Primary antibody against Akt was from Biogot (Nanjing, China). Antibodies of mTOR, MPO, CitH3 were from Abcam (Cambridge, UK).