Dulbecco modified eagle medium (dmem)

To mimic the astrocyte activation after transient cerebral ischemia in vitro, we established the OGD/R model in the primary culture of mice astrocytes according to a previous report [23 (link)]. Briefly, the primary-cultured astrocytes were rinsed twice with phosphate-buffered saline (PBS, pH 7.4), and then refreshed with glucose free DMEM (Gibco). Cells were then placed in a sealed chamber (Billups-Rothenberg, San Diego, CA, USA), loaded with mixed gas containing 95% N₂, and 5% CO₂ at 37 °C for 2–6 h. Following the insult, cultures were transferred back to a DMEM containing 10% FBS and reoxygenated in a humidified incubator (37 °C, 5% CO₂) for an additional 24 h. Cells in the normal control group remained untreated and were incubated in a regular cell culture incubator (37 °C, 5% CO₂). All astrocytes were harvested 24 h after reoxygenation.

Human proximal tubular epithelial cells (HK-2) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; ThermoFisher, Hudson, NH, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; ThermoFisher, Hudson, NH, USA). Modified oxygen and glucose deprivation (OGD) was used as a model of in vitro ischemia conditions. OGD of HK-2 cells was induced by changing the medium to serum/glucose free DMEM medium and then incubating them in a Modular Incubator Chamber (MIC-101; Billups-Rothenberg, Del Mar, CA, USA), and the chamber was flushed with 94% N₂ and5% CO₂ to maintain the oxygen concentration at 1%. After 15 h of hypoxia, the medium was replaced with fresh oxygenated medium containing serum and glucose, and the cells were returned to normoxic conditions for 30 min, 2 h or 6 h. For the hypoxic preconditioning protocol, cells were subjected to OGD for 6 h, followed by 2 h of reoxygenation before prolonged H/R injury. Cells in the normoxic group were cultured in a normoxic chamber (21%O₂) for the indicated time periods. To suppress autophagy, 5 mM 3-methyladenine (3-MA; Sigma-Aldrich, St. Louis, MO, USA) was added to the medium 1 h before each experiment.

Human renal proximal tubular epithelial cells (HK-2; ATCC, Manassas, VA, USA) were isolated and cultured in Dulbecco's modified Eagle's growth medium (DMEM; ThermoFisher, Hudson, NH, USA) containing 10% fetal bovine serum under 5% CO₂ and 95% atmospheric air at 37°C. To establish a hypoxia/reoxygenation (H/R) model in vitro, renal cells were subjected to oxygen and glucose deprivation (OGD) by changing the medium to serum/glucose-free DMEM and then were incubated in the Modular Incubator Chamber (MIC-101; Billups-Rothenberg, Del Mar, CA, USA) for 15 h in an atmosphere of 1% O₂, 5% CO₂, and 94% N₂ at 37°C, followed by reoxygenation in normal complete medium and normoxic condition for 30 min, 1 h, 2 h, 6 h, or 12 h. Transient OGD for 6 h and subsequent reoxygenation for 2 h were implemented before prolonged H/R injury to achieve HPC. Control cells were cultured under normal condition. The experimental design is detailed in Figure 1.