I have been looking at protocols for an in vitro myocardial ischemia/reperfusion model using Human Umbilical Vein Endothelial Cells (HUVEC) to look at Transforming growth factor beta 1 (TGF-β1) signalling.

Some of the protocols merely put the cells in a hypoxic chamber for 4 to 24 hours followed by a medium change. Other protocols use an ischemic buffer combined with an anoxic environment but for a shorter period. An example of an ischemic buffer: 1 mM NaH2PO4, 24 mM NaHCO3, 2.5 mM CaCl2, 118 mM NaCl, 16 mM KCl, 0.5 mM sodium EDTA, 20 mM sodium lactate, pH 6.8. Followed be a "reperfusion buffer" including glucose.

I have looked at the pathophysiology of ischemia-reperfusion injury, and believe that moving the cells to an anoxic environment without glucose supply (ischemia insult) for a time period followed by replacing the medium with oxygenated medium supplemented with glucose would suffice.

I am not sure which approach is the most accurate and would value any opinion, protocol or experience.

Thank you