I need to perform some experiments with HEPG2 cells in hypoxia conditions.
I have a doubt on the mixture of gas we have to use.
Is the mix 1%O2/94%N2/5%CO2 correct? or should I work in complete absence of oxygen?
This may seem pedantic, but it depends on what you mean by "hypoxic", and the way that you aerate and keep the cells. Many are rather loose in their interpretation of hypoxia, and it depends on how severe a challenge you want. On the one hand cells obviously consume oxygen, and adequate oxygen delivery in vitro can be a problem, but on the other hand oxygen is pretty soluble in most plasticware used for cell culture, and even with nominally zero O2 in the gas mix the actual measured PO2 in the fluid can be 10-20 mmHg just because of diffusion. Wolfgang's paper beautifully demonstrates the problem, and I completely agree with his conclusion that you should actually measure the PO2 whatever the gas mix you use, because the PO2 will differ according to conditions. In the oxygen sensing world the "physiological" values for normoxia and hypoxia have also been a source of some controversy, as different cells normally exist in vivo in quite different levels of PO2 - tumour derived cell lines are from tissues that are often quite "hypoxic" compared to others. A discussion of some of these aspects in the attached review, if it is of any help.
http://www.sciencedirect.com/science/article/pii/S0005272807002496
This may seem pedantic, but it depends on what you mean by "hypoxic", and the way that you aerate and keep the cells. Many are rather loose in their interpretation of hypoxia, and it depends on how severe a challenge you want. On the one hand cells obviously consume oxygen, and adequate oxygen delivery in vitro can be a problem, but on the other hand oxygen is pretty soluble in most plasticware used for cell culture, and even with nominally zero O2 in the gas mix the actual measured PO2 in the fluid can be 10-20 mmHg just because of diffusion. Wolfgang's paper beautifully demonstrates the problem, and I completely agree with his conclusion that you should actually measure the PO2 whatever the gas mix you use, because the PO2 will differ according to conditions. In the oxygen sensing world the "physiological" values for normoxia and hypoxia have also been a source of some controversy, as different cells normally exist in vivo in quite different levels of PO2 - tumour derived cell lines are from tissues that are often quite "hypoxic" compared to others. A discussion of some of these aspects in the attached review, if it is of any help.
http://www.sciencedirect.com/science/article/pii/S0005272807002496
Jeremy, congratulation, this is a beautiful review of yours. By the way, if you or anybody else is interested to attend the hypoxia/erythropoietin meeting in Luebeck in June 2015, you are very wellcome (http://www.physio.uni-luebeck.de/index.php?id=162)
If you are interested in the effects of oxygen, you should definitely not do experiments in th complete absence of oxygen, as anoxia and hypoxia are completly different things. I enclose a short story I have written on what hypoxia i.
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