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?

  • Hello Linh2021,

    Thank you for contacting Agilent Cell Analysis Technical Support. Below are the responses to your questions:

    Q1. We have tested glucose at10 mM and that is why it is suggested as the concentration.. It is the lower limit of saturating glucose concentration. Higher amounts of glucose may effect the biology of your sample and thus change the outcome of the assay. 

    Q2. Pyruvate is recommended in the assay media, as it is required in some cells for maximal respiration after FCCP injection.

    Please do not hesitate to contact us if you have any questions or concerns. You can reach us by replying to this email or by calling 800-227-9770, option 3, option 8.

    Best regards,

    Agilent Cell Analysis Technical Support 

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