Hypertonic Stress Response in H9c2 Cells

H9c2, a rat ventricular myoblast cell line (Shanghai Cell Bank, Chinese Academy of Science) was maintained at 37˚C and 5% CO₂ in Dulbecco's modified Eagle's medium (HyClone; Cytiva) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% glutamine. H9c2 cells were seeded onto six-well plates and incubated with culture media containing NaCl (50 mM) or mannitol (100 mM), which yielded hypertonic state, equaling 400 mOsm/l, and treated with or without ROS scavenger N-acetylcysteine (NAC; 0.5 and 2 mM). Normal culture medium (isotonic, 300 mOsm/l) served as the control for the experiment. After exposure for desired time periods, the monolayer cultures were washed with D-Hanks and the cells were removed by 0.25% trypsin.