Modular incubator chamber mic 101

Cardiomyocytes were randomly divided into seven groups: i) control; ii) SI/R; iii) carvedilol (1 μM); iv) carvedilol (5 μM); v) carvedilol (10 μM); vi) TLR4 inhibitor (20 μg/ml; an anti-TLR4 blocking antibody; CST Technologies, Boston, MA, USA) and; vii) pyrrolidine dithiocarbamate (100 μmol/l PDTC; NF-κB inhibitor) groups. In the control group, H9c2 cardiomyocytes were cultured under normal conditions in 5% CO₂ incubation. SI/R experiments were performed according to a method previously described by Esumi et al in 1991 (13 (link)). Briefly, the cardiomyocytes were exposed to ischemia by replacing the medium with an ‘ischemic buffer’ designed to simulate the extracellular environment during myocardial ischemia, with the approximate concentrations of potassium, hydrogen and lactate ions that are observed to occur in vivo [137 mmol NaCl, 12 mmol KCl, 0.49 mmol MgCl₂, 0.9 mmol CaCl₂·2H₂O, 4 mmol 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 20 mmol sodium lactate (pH 6.2)]. Cells were incubated in a hypoxic/ischemic chamber also known as the Modular Incubator Chamber (MIC-101; Billups-Rothenberg, Del Mar, CA, USA) at 37°C for 2 h in a humidified atmosphere of 5% CO₂ and 95% nitrogen. For the reoxygenation process the cells were superfused in DMEM supplemented with 10% fetal calf serum at 37.1°C under 5% CO₂ incubation for 2 h.

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.

For hypoxia treatments, A2780 cells were incubated under hypoxic conditions with 1% O₂ using a Modular Incubator Chamber MIC-101 (Billups-Rothenberg Inc., CA) or in the presence of 100 μM DFO and CoCl₂ for 24 h. Sphere cells were plated into laminin-coated 6-well tissue plates (1×10⁵/well), and CoCl₂ and DFO stock solutions were added to cell cultures to a final concentration of 100 μM. After incubation at 37°C for the indicated time periods, cell culture medium was removed, and cells were harvested after washing with HBSS solution

Neurons were exposed to H/R following the protocol as described previously [10 (link)], with a slight modification. Briefly, at the indicated time, cultured neurons were transferred to a hypoxic chamber (Modular Incubator Chamber MIC-101; Billups-Rothenberg Inc., Del Mar, CA, USA) containing 94% N₂, 5% CO₂, and 1% O₂ and incubated for 4 h at 37 °C. The culture plates were then returned to normoxic conditions (95% air and 5% CO₂ at 37 °C) and incubated for the indicated time.

HUVECs were obtained from Lonza. HUVECs were seeded on plates pre-coated with 30 μg/ml collagen type 1 and cultured in EBM medium supplemented with EBM-MV Bullet Kit (5% fetal bovine serum in endothelial cell basic medium with 12 μg/ml bovine brain extract, 1 μg/ml hydrocortisone, 1 μl/mL GA-1000. HBMEC cells were purchased from Cell System (Kirkland, WA, United States) and grown in plates pre-coated with attachment factor (Cell Systems) and cultured in serum-containing medium (Cell Systems). Both HUVECs and HBMECs at early passage (P3) and cells upon ∼70–80% confluency was used for all experiments. The cell morphology was observed under an inverted microscope. For hypoxia treatments, 70–80% confluent HUVECs and the HBMECs were exposed to 1% oxygen for 24, 48, and 72 h using the hypoxia chamber (Modular Incubator Chamber (MIC-101), Billups-Rothenberg, Inc. The Normoxia cells were treated with 20% oxygen.