I am using SH-SY5Y cells for hypoxia in a hypoxia chamber which I got from stem cell technology. I am using commercial pre mixed cylinder (2% O2, 5% CO2 and balance N2). I am using hanks balanced salt solution without glucose as a hypoxia medium. Initially it took 16 hrs incubation to kill 50% cells, average of 54% with consecutive 2 experiments. Now when I started to screen my test compounds, there were no destruction of cells in hypoxia control wells. There is no leakage in chamber. I don’t know what the problem is. There is no leakage in chamber found. Can anyone suggest what could be the problem? Whether need to change gas mixture composition? Can I use only nitrogen with HEPES in medium and eliminating CO2 and O2? as the pre mixed gas cylinder may have consistency issue after frequent usage. I will entertain other alternatives also.
Thank you.
I suppose, O2 infusion should not be used while running hypoxia experiments. So If I were you, I would stop O2 infusion and just try with (CO2 and Nitrogen) or may be try Nitrogen alone but CO2 is necessary for cells to grow. However, infusion rate of nitrogen should also needs to be monitored. Run a pilot experiment and then decided which one to choose. The possible reason behind no death in control cells could be the media itself. As dissolved oxygen in media might have rescued cells from dying. So again If I were u, I would incubate required volumes of media overnight in hypoxia chamber in sterile flat bottom cell culture flask to remove oxygen and then next day use that media for hypoxia experiments. Hope it helps!
Hi, I would check if 2% hypoxia increases ABC transporters expression in SH-SY5Y cells, knowing that these cells do have bone marrow origin and hypoxia promotes more immature phenotype in them. Most probably less than 1% O2 will be more appropriate for your experiment. Best regards, Lena
Thank you for your suggestions and information. I will do as recomended
It has been a long time since I did this stuff, but when I did it, I used to bubble hypoxic gas mixture through the medium to get it hypoxic before adding to the cells. I also used to test the PO2 of the medium by running it through a gas analyser and use to be surprised by quite how hard it was to get the PO2 down. I think it takes a long time to equilibrate if you put fresh medium in to a hypoxic chamber.
We have done combined oxygen glucose-oxygen deprivation in NT2-N neurons. Used 95% Nitrogen and 5% carbondioxide, DMEM without glucose. Do think that you need to remove oxygen completely. Vacuum was applied to the chamber for 20–25 s (20–25 inches of Hg), and the air replaced with 5% CO2 and 95% N2. This procedure was performed four times. Inside the chamber hydrogen was generated with an envelope containing palladium catalyst to remove trace amounts of oxygen. Medium was bubbled with nitrogen before the experiment (Almaas Pediatr Res 2002). With this procedure oxygen concentration was 0.1% after the fourth gas exchange, and fell to
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