Currently we culture our MDA-MB-231 cancer cells in DMEM (containing 25mM glucose) with 10% FBS + 1% PenStrep. I am wanting to assess the effects of different glucose concentrations (25 mM and 5 mM glucose) on metabolic pathways (glycolysis and b-oxidation) in these cells treated with ketone bodies. I have seen that one way of assessing low glucose concentrations in these cells would be to culture them in 25 mM glucose, glucose starve the cells and supplement with pure glucose at 5 mM before each experiment. I am worried that this would induce metabolic alterations consistent with acute hypoglycaemia and does not accurately depict long term normoglycaemic conditions! Can anyone perhaps help me with information on how I can culture these cells in 5 mM glucose without inducing metabolic alterations that will affect my experiments?
You do not have to starve the cells for glucose. You can simply use your desired conditions (5mM and 25mM) from the beginning in two separate batches of cells. Make sure that enough time has elapsed to deplete excess glucose after your initial switch of medium from 25mM to 5mM.
There may be some difference in growth rate of cells so remember to compensate for it when you compare the two conditions. Either use an equal number of cells or normalize your variables for the same number of cells.
Agreed with Tausif Alam
Prefer begin in two separate batches of cells.
see the article i hope help you .
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0108444
We try to grow the cells at low glucose concentration (5mM) for certain period of passage number then once they adapt to the low gluocse condition then we shift them to high glucose media according to experimental set up. We collect the cells at different time points and check their metabolic profile using real time approach. We also look for some inflammatory genes also. In our studies we have shown the rise in GAPDH expression with increasing glucose concentration upto certain period of time. Then its leads to cell death. Hope this information will help you.
Thank you so much for your help Tausif Alam, Saleh Alkarim and Piyush Chadasama I really appreciate all your help.
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