You can use CoCl2

Hi

I have used  simulated ischemia solution (glucose-free Tyrode solution containing 10 mM 2-deoxy-D-glucose and 10 mM sodium dithionite) for 90 min followed by 30 min of reperfusion with the normal Tyrode solution.

 Hi Guillaume,

Have you ever measured the level of cell death in your protocol? I'm considering starving the cells of oxygen and depriving them of glucose. I need to compare the differences between ischemic-preconditioned and non-preconditioned cells so there has to be enough cell death between the two groups to see a noticeable change.

Cheers,

Sam 

Of note, dithionite ion has strong affinity to bi- and trivalent metal cations. Not sure how one might control for this and whether or not this will confound your results… As mentioned above, you can use CoCl2 at a final concentration of 100μM followed by wash(thorough) and incubation with media ( without CoCl2) as a means for "reperfusion". Good luck

HI Sam ,

In our study we were preconditioning the cells with Zinc ( and its ionophore).  I have attached the file you can see that we also mesured cell viability. 

Hello! running an hypoxia lab myself, I would say the specificity of the question you're addressing here should be run under hypoxia (1% O2) conditions... Understandably, these systems are not available to all labs, and it's common praxis to use chemical inducers of hypoxia, and depending on your query and pathways involved you can be more or less selective of the hypoxia-mimicking agent used.

To sum the mimicking agents you have been suggested here CoCl2 (100-200 microM) is your best option; not only will it stabilize molecular hypoxia (hypoxia-inducible factors) but also push the mitochondria into an hypoxia-like status. Quelating agents such as dithionite have the downfall of quelate all cations unspecifically (meaning you might be activating/inhibiting all cation-dependent pathways). I would suggest for hypoxia studies the use of deferroxamine or 2,2'-dipyridyl (100-200 microM), for both agents are more selective to divalent cations and particularly iron (which is hypoxia relevant). If your question is specifically regarding HIF-mediated hypoxia, I would suggest dimethyloxallyl Glycine (same final concentration); it specifically inhibits deoxygenases, the enzymes that recognize molecular oxygen in the cells. Finally, mind that the wash-through of these substances can be up to 6h, which means you have lost your hypoxia-inducible factors (which lasts a few minutes); this doesn't mean that your conditioning isn't in place for HIF-mediated proteins have much longer half-lives. Consider a small pilot to check which of these agents would be best in your system (they are easily accessible and fairly inexpensive).

One final remark: do you happen to have a stem-cell/differentiation lab in your vicinity? They usually have low-oxygen cell incubators. From what I gathered from your question anything between 2-0.5% O2 should would for your (i would recommend a minimum of 6h, max of an overnight, exposure to hypoxia to make sure you're properly activate both mitochondria and nucleus to respond to hypoxia).

God luck!

Careful with Cobalt tho as it effects voltage activated channels, important in excitable cells like cardiomyocytes, not sure about myoblastic h9c2s.  We tend to use BD anaerobic bags for set time, +/- glucose lactate, etc dependent on goal/endpt -hypoxia simulated ischemia - Can also use the palladium catayst system in just a aritight jar.