I am planning an lab study on effect of hypoxia on glucose metabolism of cancer cell.
For hypoxia exposure, I am considering anaerobic chamber system.
My concern is that if I use this chamber, the cells have to be exposed to room air after pulling out of the anaerobic chamber and before being put back into the incubator (probably 10-15 min).
Would effect of hypoxiabe neutralized, if the cells (possibly cancer cell such as PC9) were exposed to room air for short while? I read a study result which showed that product such as HIF can be greatly influenced from such factor.
Is there any preventive measure I can refer to?
I hope the description is detailed enough to deliver necessary information. Any advice or comment is welcome.
Thank you.
Exposing your cells to room air for 10-15min will lead to a significant portion of HIF-1a being degraded, as the half-life of HIF-1a in normal air is about 5 min. There will possibly be changes to other proteins/mRNAs etc but it's hard to know without measuring or testing this. We overcome the issue of degradation by immediately lysing cells for RNA and protein and placing straight on ice for processing. I would recommend processing the cells straight after removing from your hypoxia chamber for the best results.
Hi, Jeong
I don't know exactly what you want to analise after the hypoxia period, but if it includes HIF1a expression, you can also try to apply a drug that simulates the hypoxia effect by stabilizing HIF1a (inhibits its degradation). This also works if you are mostly interested in HIF1-mediated effects on your cells. The prolyl hydroxylase inhibitor dimethyloxaloylglycine (DMOG) is used for this purpose, and you can use it as a positive control to check if it alters the parameters you want to analise in your cells. Cobalt chloride is also used as inducer of hypoxia-like responses and you can play around with this substances depending on which parameters you are interested to check.
If you want to evaluate other proteins after the hypoxia period, HIF1a may not be present anymore, but some effects of the hypoxia (and HIF1a-mediated alterations) may be maintained. Some studies have demonstrated that 1h of hypoxia exposure is enough for astrocytes in cell culture to increase gene expression of HIF1a-modulated proteins. I checked this effect 24h after 1h-hypoxia exposure and the modulation of the parameters I wanted to see was still detectable by RT-PCRq. So, even if the cells are exposed to air room, the hypoxia effects may not be completely annulled, depending on what you are looking for. Also, you have to see if it's of interest to evaluate the cells after this 'reperfusion' paradigm. If not, prolonging your hypoxia time and analising it right away after taking them out of the chamber would be the best option.
@ Kristina M Cook
Thank you very much for your advice . Following your advice, we are designing the study so as to minimize the exposure. Thanks a lot.
@ Yasmine Nonose
We haven't made full decision on which metabollite we will be focusing on. One more question, would checking CXC4R be helpful? I read this study result showing that CXC4R can be a indirect mesurement of how much cells were exposed to hypoxia.
Jeong Uk Lim
I've never measured this CXCR4 receptor before, but I used similar controls in my studies to check if the exposure to hypoxia was being effective (including measuring MCT4 expression, which is increased by HIF1a). Also, another thing I did to make sure the chamber environment was truly hypoxic was to measure the oxygen dissolved in cell media with an oxymeter.
It seems to me that measuring CXCR4 is a good proposal, specially if you are still trying to stablish how long you should perform the hypoxia. Good luck!
@ Yasmine Nonose
Thank you once again for your kind explanation.
I hope everything goes well with your work too. Have a good day!
Jeong Uk Lim,
Transient changes in atmosphere due to opening the incubator door and transfer outside of the incubator will seriously disrupt your hypoxia-sensitive measures. If you have any concern for transient changes inside or outside of the incubator just ditch plates, dishes and flasks. These are open containers and unsuitable for high quality hypoxia studies. Just buy the Petaka G3. It is a closed cell culture cassette that automatically and precisely regulates to low internal O2 levels. Since it is functionally closed it will provide up to several hours of work outside of the incubator or glove box without significantly altering pO2 levels. Plus, it is the only cell culture technology that allows you to measure and monitor real-time pO2 levels in your culture. SO whatever you are measuring you will be able to correlate those results very carefully to the actual pO2 levels available to your cells while in culture. All in a low cost plastic disposable. www.celartia.com
Useful link please review this link:
Article Hypoxic Conditioning as a New Therapeutic Modality
Inside the hypoxia chamber itself, before your experiment, put in an air tight box. After your experiment is done, before bringing your cells back out into normoxia, instead put your cells into the air tight box and seal it. Now when you take them into the normoxic environment, the box microenvironment is still hypoxic, and so are your cells.
- Badges
- Science topic
- Similar topics
- Medicine
- Oncology
- Cancer Research
- Cancer Biology
- Cancer Cells