I am beginning to perform several experiments using cancer cells in a hypoxic environment. Our core facility has a hypoxic incubator that uses nitrogen to displace oxygen down to a minimum of 1% O2.
However, my cells make take up to a week to differentiate and in that time lactic acid production from the cancer cells (Warburg effect) acidifies the media, so I will need to change it once or twice during my experimental paradigm.
How can I perform this with minimal O2 perturbations?
I understand that without substantial $$$ for a hypoxic chamber I can't completely prevent reoxygenation, but does anyone have any low cost solutions/ideas for managing these tasks?
Currently the front-runner idea is to maintain my media in the hypoxic chamber and fill a large gas-tight box with N2 to displace the O2 and perform media changes inside that box, but that seems a little slapdash.
Dear Levi Adams,
In my case I am using DMOG is a cell permeable prolyl-4-hydroxylase inhibitor. To mimic Hypoxia instead of Hypoxia chamber (stem cell technologies). DMOG has the same effect compared with H. chamber.
Hopefully this information useful for you.
Hamad
Hi Levi, we use a glass bottle with a special cap to run N2 gas through the cell culture medium for a couple of hours. It's a cap with a long tube that bubles N2 into the medium and a shorter tube for exit-gas, both with a filter to keep it sterile. Could that work for you?
And have you thought about adjusting the buffer capacity of your media to compensate for the lactic acid production? Or using an addition buffer like HEPES?
Hi Levi
there is a whole series of papers by Greg Semenza’s lab describing how to maintain cells in hypoxic conditions. One crucial point is to add HEPES buffer at 25 mM in your media to avoid acidification which will affect your experiments in f you expose your Cell to hypoxia for more than 24 hours.
See for instance this paper
Article Analysis of Hypoxia-Induced Metabolic Reprogramming
Francesca
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