Abdfacs canto 2 flow cytometer

Lung tissue samples collected from healthy newborn male piglets (Tibetan pigs and Landrace pigs) at 7 days of age were soaked in PBS. Primary ATII cells were isolated as described previously (Wang et al., 2016 (link)) with minor modifications. Then, ATII cells were cultured in complete medium at 37°C in an environment containing either 21% O₂, 5% CO₂, and 79% N₂ (normoxic conditions) or 2% O₂, 5% CO₂, and 98% N₂ (hypoxic conditions).
We harvested ATII cells (n = 6 for each group) at 48 h under the 21% O₂ (Tibetan–normoxic (TN) and Landrace–normoxic (LN)) or 2% O₂ (Tibetan–low hypoxic (TL) and Landrace–hypoxic (LL)) conditions. Three of each group were flash-frozen in liquid nitrogen for RNA extraction, and the rest were used for analyze cell apoptosis by A BD FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA).

Caspase-8 and caspase-9 activitiesweremeasured using the FLICA Caspase-8 Assay Kit and FLICA Caspase-9 Assay Kit (ImmunoChemistry Technologies, Bloomington, MN, USA), respectively, according to the manufacturer’s protocol. In brief, MCF-7 and MDA-MB-231 breast cancer cells (1 × 10⁶) were washed with cold PBS twice and re-suspended in buffer. Then, 5 μL of diluted FLICA reagent and 2 μL of Hoechst 33,342 were added to 93 μL of cell suspension, mixed by pipetting and incubated for 60 min at 37 °C. After incubation, the cells were washed twice with 400 μL of apoptosis wash buffer and centrifuged at 300× g. After the last wash, the cells were resuspended in 100 μL of apoptosis wash buffer supplemented with 10 μg/mL of PI. Analyses were performed using aBD FACSCanto II flow cytometer, and the results were analyzed with FACSDiva software (version 6.1.3, BD BiosciencesSystems, San Diego, CA, USA). To identify the involvement of caspases in apoptosis induced by compounds 1–3, the investigated breast cancer cells were pretreated with the pan-caspase inhibitor, Z-VAD-FMK (100 µM) (Sigma Aldrich, St. Louis, MO, USA), as described in [28 (link)]. The activation of caspases-8 and -9 was detected as described above.