Seahorse Cell Mito Stress Medium

For preparing seahorse XF cell mito stress test assay media, the Agilent's protocol suggests adding 1 mL 100mM pyruvate solution (final pyruvate conc = 1mM), 1 mL 200 mM glutamine solution (final glutamine conc = 2 mM) and 1 mL 1.0 M glucose solution (final glucose conc= 10mM) in 97 mL XF base media. However, we use 25mM glucose (4.5 g/L) DMEM medium which does not include pyruvate for fibroblast culture maintenance. Q1: The recommended glucose concentration is 10mM in the Agilent’s protocol, but we regularly use 25mM glucose concentration for our cell culture, will higher concentration of glucose interfere with the assay performance or machine detection? Q2: Also, our regular cell culture media do not contain pyruvate. If we do not add pyruvate into the seahorse media, will pyruvate significantly limit the rates of respiration and/or extracellular acidification?

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