During Seahorse mitostress assays, we have great variance between different cell lines regarding their oxygen consumption. One cell line is tricky because at relevant confluency, the basal OCR is quite high, and then it gets very high after adding FCCP and correspondingly the oxygen level in the wells drops dramatically and has been down to between 10 and 20. How low can the O2 level be without having hypoxia? I realize that if we get hypoxic conditions during the run, the OCR values will get a "false" value that is lower than it should be because the cells have not enough O2 to consume. I'm just unsure when this kicks in - is it around 20, 10 or 5 mmHg?
FCCP uncouples ATP resynthesis from oxygen consumption. If cellular respiration is fully uncoupled, you get no ATP production regardless of the O2 level in the bathing media or the rate of O2 consumption. In that situation, ATP can only be resynthesized anaerobically (i.e., via anaerobic glycolysis or reaction of phosphocreatine with ADP) and that can go on for only a relatively brief period of time. When those sources of ATP are depleted, death ensues.
This will happen with hearty cells/isolated mito when OCR exceeds around 500 pmol O2/min or so (in the XF96). You can usually notice this happening when you see your O2 level data starts to look like a backwards 'J'. This is nicely described with 'Fig 7' from the following protocol article about respirometry in frozen mitochondria samples.Article Measuring Mitochondrial Respiration in Previously Frozen Bio...
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