Dulbecco s minimal essential medium

MycOE Pik3ca Mut cells, MycOE Pten Null cells and mouse embryonic fibroblasts were cultured in Dulbecco's minimal essential medium (Cellgro) supplemented with 10% (vol/vol) fetal bovine serum (FBS), 100 IU penicillin, 100 μg/ml streptomycin (Cellgro) and 2 mM l-glutamine (total 6 mM l-glutamine) (Cellgro). HEK293 and HeLa cells were cultured in Dulbecco's minimal essential medium (Cellgro) supplemented with 10% (vol/vol) fetal bovine serum (FBS), 100 IU penicillin, 100 μg/ml streptomycin (Cellgro). DBTRG-05MG and HCT116 cells were cultured in RPMI-1640 supplemented with 10% (vol/vol) FBS, 100 IU penicillin and 100 μg/ml streptomycin.

Trigeminal ganglia and DRG cell cultures were prepared as previously described (Lee et al., 2018 (link); Tonello et al., 2019 (link)). Briefly, mice were terminally anesthetized with isoflurane, and TGs or lumbar, thoracic, and cervical DRGs were bilaterally removed aseptically from 8-week-old mice. Removed TG or DRG tissues were incubated in papain (60 U, Cat# P3125, Millipore Sigma) for 20 min at 37°C followed by collagenase (1 mg/ml, Cat# C6885, Millipore Sigma) for another 20 min at 37°C. Tissues were mechanically dissociated, and cells were then cultured in Dulbecco’s Minimal Essential Medium (Cat# 15017CV, Corning Inc., Corning, NY, United States) completed with 10% fetal bovine serum and 1% pen/strep in eight-well chambered cell culture slides (Cat# 354118, Corning) precoated with a cell and tissue adhesive (Geltrex, Cat# A1569601, Thermo Fisher Scientific). Cultures were incubated at least 24 h at 37°C with 5% carbon dioxide before each experiment. These cultures were treated with PBS vehicle control or TRPC4 agonist Englerin A (100 nM to 10 mM) with or without ML204 (100 μM) for 24 h and then culture media were collected and stored at −80°C for further ELISA analyses.