Hypoxia not up-regulating HIF-1a. Need help. Thanks. - (Sep/07/2011 )
We have been using our new hypoxia incubator by growing mesenchymal stem cells or human chondrocytes in it at 2% Oxygen level. It is the type that uses nitrogen gas to expel the oxygen. We have so far had no luck in seeing any HIF-1a or HIF-2 upregulation using western blotting. Also, we have been doing qPCR on multiple genes that have a HIF responsive element such as collagen 2, aggrecan and hyaluronic acid synthase and seeing no increase in them either.
Does anyone have any advice on troubleshooting this problem? It is quite comical how similar 2% oxygen and 20% oxygen cells are in every way.
2% oxygen is quite similar to the physiological oxygen levels in tissue (which is ~3%), so I wouldn't be surprised if you have no or low induction of hypoxia-related genes. We use 1% O2 in our lab, but I'm not a specialist...
1% O2 is a good level. How long did you keep them in hypoxia?
Many people see upregulation in HIF at 5% o2. We did not so we lowered it to 2% o2. Good idea though we can try going even lower. Our experiments are usually 14 day pellet culture in hypoxia.