Primary human renal proximal tubular epithelial cells (RPTECs) (cat. no. 4100, ScienCell, Carlsbad, CA, USA), primary Syrian hamster RPTECs (cat. no. HM-6015, Cell Biologics, Chicago, IL, USA) and primary C57BL/6 mouse RPTECs (cat. no. C57-6015, Cell Biologics) were cultured in Complete Epithelial Cell Medium/w kit (cat. no. M6621, Cell Biologics), supplemented with epithelial cell growth supplement, antibiotics, and fetal bovine serum. All the above primary cells were differentiated, well-characterized passage one RPTECs. We expanded them in 75 cm2 flasks and, consequently, passage two cells were used for the experiments.
Cells were cultured in 6-well plates at a number of 300,000 cells per well, or in 96-well plates at a number of 10,000 cells per well, for 16 h, before the onset of anoxic conditions. The confluency of the cells, as estimated by inverted microscopy, did not differ at the start of each experiment. The GasPakTM EZ Anaerobe Container System with Indicator (cat. no. 26001, BD Biosciences, S. Plainfield, NJ, USA) was used to reduce oxygen levels to less than 1%. Cells within the anaerobe container were cultured at 37 °C. These anoxic conditions imitate warm ischemia.
Cell photos were captured at the onset of hypoxia and at 2-h intervals. For this purpose, an inverted microscope (Axiovert 40C, Carl Zeiss Light Microscopy, Göttingen, Germany) and a digital camera with the related software (3MP USB2.0 Microscope Digital Camera, Amscope, Irvine, CA, USA) were used.
Imaging of each cell type was used to detect the approximate time of severe cell deterioration (death) due to anoxia. Reperfusion experiments were started at a point corresponding to half of this time. In these experiments, cells were washed, supplemented with fresh culture medium, and placed at 37 °C in a humidified atmosphere containing 5% CO2. These reoxygenation conditions imitate warm reperfusion. The time point of severe cell deterioration due to reoxygenation was also detected with imaging.
As live cells were required for conducting the experiments, the various parameters of the study were evaluated at the halfway point of the time needed for detecting severe cell deterioration, with cell imaging under anoxia or after 2 h of reoxygenation. The same time points for mouse and hamster cells were used, since the latter showed remarkable resistance to cell death by anoxia. All the experiments were performed 9 times.
Cells were cultured in 6-well plates at a number of 300,000 cells per well, or in 96-well plates at a number of 10,000 cells per well, for 16 h, before the onset of anoxic conditions. The confluency of the cells, as estimated by inverted microscopy, did not differ at the start of each experiment. The GasPakTM EZ Anaerobe Container System with Indicator (cat. no. 26001, BD Biosciences, S. Plainfield, NJ, USA) was used to reduce oxygen levels to less than 1%. Cells within the anaerobe container were cultured at 37 °C. These anoxic conditions imitate warm ischemia.
Cell photos were captured at the onset of hypoxia and at 2-h intervals. For this purpose, an inverted microscope (Axiovert 40C, Carl Zeiss Light Microscopy, Göttingen, Germany) and a digital camera with the related software (3MP USB2.0 Microscope Digital Camera, Amscope, Irvine, CA, USA) were used.
Imaging of each cell type was used to detect the approximate time of severe cell deterioration (death) due to anoxia. Reperfusion experiments were started at a point corresponding to half of this time. In these experiments, cells were washed, supplemented with fresh culture medium, and placed at 37 °C in a humidified atmosphere containing 5% CO2. These reoxygenation conditions imitate warm reperfusion. The time point of severe cell deterioration due to reoxygenation was also detected with imaging.
As live cells were required for conducting the experiments, the various parameters of the study were evaluated at the halfway point of the time needed for detecting severe cell deterioration, with cell imaging under anoxia or after 2 h of reoxygenation. The same time points for mouse and hamster cells were used, since the latter showed remarkable resistance to cell death by anoxia. All the experiments were performed 9 times.
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