For hypoxia/reoxygenation (H/R) studies, confluent HK2 cells were incubated under a hypoxic condition of 1% O2, 94% N2, and 5% CO2 in a controlled hypoxic chamber (Thermo Scientific) for 24 h. Then, they were removed to a normoxic condition of 21% O2, 74% N2, and 5% CO2 for reoxygenation. Controls were cultured under a normoxic incubator for identical times. HK2 cells were treated with ATP (Sigma-Aldrich) at indicated concentrations for 8 h. Apyrase (0.2 U/ml, 1 U/ml, or 5 U/ml) or A438079 (20 µM) was added to the culture medium for 1 h before exposing to ATP or H/R. Experiments were performed in triplicate.
Controlled hypoxic chamber
Manufactured by Thermo Fisher Scientific
The controlled hypoxic chamber is a laboratory equipment designed to provide a controlled low-oxygen environment. It is used to simulate hypoxic conditions for various experimental applications.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
N acetylcysteine nac, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium f12, by Thermo Fisher Scientific (1 mentions)
Tcmk 1, by Merck Group (1 mentions)
Tcmk 1, by American Type Culture Collection (1 mentions)
2 protocols using controlled hypoxic chamber
1
Hypoxia-Reoxygenation Effects on HK2 Cells
2
Hypoxia-Reoxygenation and Oxidative Stress in Kidney Cells
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The TCMK-1 (murine tubular epithelial cells) and human kidney 2 (HK2) (proximal tubular cells) cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, VA). Cells were cultured in Dulbecco's modified Eagle's medium/F12 (Thermo Scientific, Rockford, IL) supplemented with 10% fetal bovine serum (Thermo Scientific) at 37 °C in 5% CO 2 . For H/R research, confluent TCMK-1 or HK2 cells were incubated under hypoxic conditions (1% O 2 , 94% N 2 , and 5% CO 2 ) in a controlled hypoxic chamber (Thermo Scientific) for 24 h, and then removed or not to a normoxic incubator for reoxygenation. Normoxic controls were cultured under normoxic conditions (21% O 2 , 5% CO 2 , 74% N 2 ) for identical times. For the H 2 O 2 study, confluent TCMK-1 cells were exposed or not to H 2 O 2 at different doses for 24 h. In certain experiments, cells were pre-administered different doses of recombinant Klotho protein or 30 µM N-acetylcysteine (NAC; Sigma-Aldrich) for 1 h.
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