The rat alveolar macrophage cell line NR8383 (American Type Culture Collection) was maintained in F12 medium (Thermo Fisher Scientific, Inc.) supplemented with 15% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 µg/ml streptomycin, 100 U/ml penicillin and 2 mmol L-glutamine (Beyotime Institute of Biotechnology) at 37°C in a humid atmosphere of 5% CO2. The medium was discarded and replaced by fresh medium every 3 days until the NR8383 cells reached 60% confluence. Before passaging, adherent cells were harvested, and then centrifuged (100 × g, 4°C, 5 min) and transferred to new microplates.
For induction of ALI in vitro, the cells were stimulated with or without 1 µg/ml LPS for various times (6, 12, 24 and 48 h) at 37°C. Short hairpin (sh)RNA targeting salusin-β and HO-1 together with shRNA control were designed and synthesized by Shanghai GenePharma Co., Ltd. Then, 20 µg pcDNA 3.1 plasmids (Thermo Fisher Scientific, Inc.) and 20 nM shRNAs were transfected into cells at 70–80% confluence using Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions, as described previously (23 (link)). At 48 h post-transfection, cells were selected for subsequent experiments.
For induction of ALI in vitro, the cells were stimulated with or without 1 µg/ml LPS for various times (6, 12, 24 and 48 h) at 37°C. Short hairpin (sh)RNA targeting salusin-β and HO-1 together with shRNA control were designed and synthesized by Shanghai GenePharma Co., Ltd. Then, 20 µg pcDNA 3.1 plasmids (Thermo Fisher Scientific, Inc.) and 20 nM shRNAs were transfected into cells at 70–80% confluence using Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions, as described previously (23 (link)). At 48 h post-transfection, cells were selected for subsequent experiments.
