Mice were sacrificed by cervical dislocation after deep isoflurane-induced anesthesia (4% for induction). Their LGs were cut into small fragments (1 mm3) and treated with MEM-α and 0.1% type 2 collagenase under shaking on a shaker at 50 rpm at 37°C for 30 minutes. After blowing and mixing, as well as centrifuging at 1000 rpm for 5 minutes, the precipitate was uniformly dissolved in Dulbecco's modified Eagle's medium containing 20% fetal bovine serum. The supernatant was discarded again, and the resulting precipitate was fully mixed with Dulbecco's modified Eagle's medium containing 20% fetal bovine serum.
We treated the primary LG epithelial cells with MCC950 (10 µM/mL; TargetMol) for 24 hours in order to inhibit the NLRP3 inflammasome. For antioxidation experiments, the primary LG epithelial cells were treated with an N-acetyl-L-cysteine (NAC) antioxidant (1 mM; Sigma, St. Louis, MO, USA) for 24 hours. Thereafter, the treated primary LG epithelial cells of passage 2 were collected for Oil Red O (ORO) staining, JC-1 staining, TUNEL staining, and Western blotting.
We treated the primary LG epithelial cells with MCC950 (10 µM/mL; TargetMol) for 24 hours in order to inhibit the NLRP3 inflammasome. For antioxidation experiments, the primary LG epithelial cells were treated with an N-acetyl-L-cysteine (NAC) antioxidant (1 mM; Sigma, St. Louis, MO, USA) for 24 hours. Thereafter, the treated primary LG epithelial cells of passage 2 were collected for Oil Red O (ORO) staining, JC-1 staining, TUNEL staining, and Western blotting.