Imark microplate spectrophotometer

In a pilot study, cell viability was evaluated after culture under 21%, 10%, 5%, or 1% O₂ for 0, 24, 48, 72, or 96 h, with the Cell Proliferation Kit I (Roche, Indianapolis, IN, USA), according to the manufacturer’s instructions. The optical density (OD) of each well was measured at a wavelength of 570 nm (OD₅₇₀) using an iMark microplate spectrophotometer (Bio-Rad, Hercules, CA, USA). Cell viability was determined as (OD₅₇₀ treated cells/OD₅₇₀ untreated cells at time 0) × 100. A long-term culture of cells under 5% O₂ was then established based on the pilot study data (S2 Fig). At 30 and 90 days after culture under 21% or 5% O₂, cell viability was evaluated as previously described.

Liver TG were determined using the EnzyChrome triglyceride assay kit (BioAssay Systems, Hayward, CA, USA). Tissue (10 mg) were homogenized in 100 μL of 5% Triton X‐100 and incubated in water bath for 5 min in which the temperature increases from 80 to 100 °C. The procedure was repeated three times, allowing the samples to settle at 20 °C between cycles. Samples were then centrifuged (16 000 g for 5 min at 4 °C), the supernatant was diluted 1 : 10 in MilliQ water (Millipore, Burlington, MA, USA) and 10 μL of this was used for the assay performed in triplicate in 96‐well plates. Samples were mixed with 100 μL of kit working reagent and incubated (15 min at 20 °C) before plates were read at 595 nm wavelength in a Bio‐Rad iMark microplate spectrophotometer.

Liver and serum triglycerides were determined using the Enzyme Chrome triglyceride assay kit (BioAssay Systems, Hayward, CA, USA). Tissue (10 mg) were homogenized in 100 µL of 5% Triton X-100 and incubated in a water bath for 5 min in which the temperature rose from 80 °C to 100 °C. The procedure was repeated three times, allowing the samples to settle at 20 °C between cycles. Samples were then centrifuged (16,000× g, 5 min at 4 °C), the supernatant was diluted 1:10 in Milli-Q water, and 10 µL were used for the assay performed in triplicate in 96-well plates. Sera were diluted 1:5 in Milli-Q water and 10 µL were used for the analysis performed in triplicate in 96-well plates. Samples were mixed with 100 µL of kit working reagent and incubated (15 min at 20 °C) before the plates read at 595 nm wavelength in a BioRad iMark microplate spectrophotometer.