Hypoxia Treatment For Cell Culture - (Nov/23/2009 )
hi, i was just wondering if anyone knows how long you can treat cells with CoCl2 or deferoxamine to induce hypoxia before it becomes toxic...i am sure it is different for a given cell type, but just wanted to get a general idea...also, what concentrations you would recommend (as of now, i am set on using 100uM for both). thanks =)
Somewhere between 50 and 200ÁM CoCl2 should work. I have not done longer than 24h treatment - but I wouldn't recommend going much longer than that either.
While both DFO and CoCl2 get you stabilization of HIF1alpha protein, it is not the only thing that they do.. this means you might be chasing ghosts if you overdo that.
Hypoxia chambers (like from Billups-Rothenberg) are not so expensive and quite easy to use.. for longer term experiments I would probably use that instead of DFO/CoCl2
hey warsel, thank you very much for the help! if i were to use a hypoxia chamber, what kind of gas mixture would i need? would it consist of just oxygen and nitrogen or would there be some CO2 as well? i was thinking somewhere along the lines of 5% O2 for inducing hypoxia...just not sure what else i need to fill up the rest of the space.
You'll need some CO2 to balance the pH of the medium. Use as much as you would in a normal incubator (5 or 10%) The rest can be made up with nitrogen. You may be able to buy gas mixes designed for this sort of work from BOC.
I used 1% Oxygen, 5% CO2, rest N2 - I got the gas pre-mixed and it wasn't overly expensive.
You could probably go 5% CO2 rest N2, but I somehow liked the defined hypoxia gas better than the complete anoxia.