We are using hypoxia chambers to induce physiological hypoxia in cell cultural environment. the major problem is, reoxygenation during the cell harvesting, cell lysis, etc.
this is the chamber we use,
http://www.brincubator.com/hypoxiachamber.htm
Do any one suggest a way to avoid the reoxygenation?
If you are removing cells from the hypoxia chamber there is no way to avoid reoxygenation to some extent. You could leave the cells in the hypoxia chamber and move them to an anaerobic chamber where cell harvesting can be performed. You could also potentially use chemicals for oxygen scavenging but I am not sure how this will affect downstream applications. Good luck.
If you just want to get the cell lysate, proteins could be extracted from cells within 10 s by rapidly removing the medium and scraping the cells into SDS lysis buffer. Otherwise, as Rick Alleman said, you can use an anaerobic workstation, however, it costs a lot. Good luck!
Firstly, that isn't a hypoxic chamber. Although it may be sold as a hypoxic chamber, it isn't. The way to know is if you place either a solution of oxyhemoglobin or blood in there and observe the color change. Better yet use anaerobic indicator strips which are sold by Oxoid.
Assume that you do not have hypoxia until you can prove that you don't.
Secondly, the only way to do what you are suggesting is to use a glove-boz and make sure all your reagents are oxygen free, then fix the cells anaerobically.
Your best bet for hypoxia might be to use dense cultures; i.e., spin the cells to the bottom of a glass (not plastic, because it holds and leaches O2) centrifuge tube and then incubate them in your CO2 incubator. The surface cells (up to 100 um in) will be aerobic, but the inner cells in a clump that is a mm thick, will be hypoxic. Look up the papers by Azzam EI, Little JB. The radiation-induced bystander effect: Evidence and significance. Hum Exp Toxicol 2004; 23: 61-65. The technique they used also provides hypoxic cells when many cells are used.
- Badges
- Science topic