Hi, I am using bone marrow derived macrophages (untreated). I have tried different seeding densities however I do not see a significant increase in ECAR after glucose (25mM) addition. How can I fix that? We used XFe 96, and the glycolysis stress test. Cells were differentiated in 20% L929 for 7 days and cultured in high glucose DMEM. Cell media was changed an hour before experiment to seahorse DMEM medium without FBS and no glucose.
Thanks!
First you answer a quiry. Do you incubate the cells in glucose free basal DMEM provided by the company for 1 hour prior to incubation ? It might be due to if the cells are not activated, then they wont be metabolically that much active. What is the proliferation rate of the cells ?
They are primary cells, bone marrow derived macrophages and non proliferative. the cells were washed twice with medium and incubated for 1hr in glucose free DMEM from Agilent in a non CO2 incubator.
Generally the highly proliferative cells like cancer cells show higher increase in ECAR after gluvose injection. As these are primary non proliferative cells, they are maight be utilizing only a small amount of glucose for their respiration. Even if the pyruvate is channeled to the TCA cycle instead of lactate. That will cause low lactate production and medium acidification rate.
In your data there is small increase in ECAR after glucose injection, which cell concentration dependent.
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