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Cardiomyocytes are usually cultured under normoxic conditions, but a variety of injury models result in hypoxia and demonstrate that the relationship between cell function and the redox state is complex. Hypoxic cultured cardiomyocytes are a popular in vitro model used in studies of hypoxia-induced cardiomyocyte dysfunction and death. The present study determined the concentration of agents that could deplete oxygen without affecting cell viability and cellular function, including sodium salicylate, chromium dioxide, diethyl pyrocarbonate, 0.005% hypoxanthine, and 0.025% sodium azide. The mitochondria were also the primary organelle to produce the reactive oxygen species (ROS). The production of ROS was measured with dihydrorhodamine (DHR), and the mitochondria activity was evaluated by rhodamine 123 (Rh 123) using flow cytometry. The results show that 0.025% sodium azide at 1 h was the most effective agent to deplete oxygen and was less toxic to the cells. In comparison, 0.005% hypoxanthine and diethyl pyrocarbonate were the next two most effective agents to deplete oxygen at early stages but 0.025% sodium azide was more effective over longer periods. Some agents have less toxic side effects at certain concentrations. The present study provides better insights into the effects of hypoxia and ROS production in hyp
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