i want to determine viability of cancer cells under hypoxic condition. i want to know that after trypsinization of cells growing under aerobic condition, can i plate the cells and incubate immediately under hypoxic condition for about 24 to 48 hrs or should allow cells to get attach under aerobic condition and then transfer the plates in hypoxic condition? After the incubation period, when MTT containing media is added, whether the plates should be incubated under aerobic or hypoxic condition?
Hi Paromita, after cell plating, the cells should be left under normal condition for at least overnight. Then incubate them under hypoxia (1% or 2% O2) until the time you want to determine how many cells survive.
MTT assay uses mitochondrial activity to convert MTT into formazan, but mitochondrial activity is decreased under hypoxia. Thus MTT assay is not suitable for determining cell viability/cell survival rate after hypoxia. Although the assay can be performed under normoxic condition after ending of hypoxic incubation, however, reoxygenation may increase intracellular levels of superoxide which directly reduce MTT to formazan leading to misinterpretation. See in Toxicol In Vitro. 2011 Dec;25(8):2147-51.
Other assays which do not depend on mitochondrial activity, such as crystal violet staining, SRB staining, and trypan blue dye exclusion are more suitable for hypoxic cells. These assays are performed at the end of hypoxic incubation period, no further incubation requirement. I have used crystal violet staining to determine effect of compounds on hypoxic survival of cancer cells, it is easy and works well. See in "A flavonoid chrysin suppresses hypoxic survival and metastatic growth of mouse breast cancer cells" Oncol Rep. 2013 Nov;30(5):2357-64.
Good luck :)
Cells need to be split at least 24 h before subjecting to hypoxia. After the treatment, the assay for viability needs to be under hypoxic condition.
Hypoxia should be induced after the attachment and continued till the assay is complete (that includes incubation with MTT and subsequent steps)
Paromita, If I am in your situation I would totally do different combinations.
Expose cells directly to Hypoxia after detachment with Trypsin (? - What is the %of Hypoxia - if you are aiming at less than 1% you may want to give your cells some time to recover from trypsination)
MTT is quite a quick assay, I would recommend doing it in both ways as mentioned above by other scientists or add MTT after your hypoxia treatment and let the cells sit with MTT in normal conditions... and study the difference.........
Good Luck and I would suggest - play with different conditions - methodology changes from exp to exp
Some cells are more sensitive to hypoxic conditions than others, make sure you have controls at non hypoxic conditions and at hypoxic conditions without any other treatment.
Hi Paromita, after cell plating, the cells should be left under normal condition for at least overnight. Then incubate them under hypoxia (1% or 2% O2) until the time you want to determine how many cells survive.
MTT assay uses mitochondrial activity to convert MTT into formazan, but mitochondrial activity is decreased under hypoxia. Thus MTT assay is not suitable for determining cell viability/cell survival rate after hypoxia. Although the assay can be performed under normoxic condition after ending of hypoxic incubation, however, reoxygenation may increase intracellular levels of superoxide which directly reduce MTT to formazan leading to misinterpretation. See in Toxicol In Vitro. 2011 Dec;25(8):2147-51.
Other assays which do not depend on mitochondrial activity, such as crystal violet staining, SRB staining, and trypan blue dye exclusion are more suitable for hypoxic cells. These assays are performed at the end of hypoxic incubation period, no further incubation requirement. I have used crystal violet staining to determine effect of compounds on hypoxic survival of cancer cells, it is easy and works well. See in "A flavonoid chrysin suppresses hypoxic survival and metastatic growth of mouse breast cancer cells" Oncol Rep. 2013 Nov;30(5):2357-64.
Good luck :)
thanks Kriengsak,
i just want to know that in your experiments , the cells have be treated with test compound for 30 minutes under normoxic condition, then it has been incubated under hypoxic condition. If a just treat the cells with the compound and immediately incubate under hypoxic condition. will there be any difference?
Hi Paromita,
In the Oncol Rep 2013 paper, the test compound was incubated with cells for 30 min before subject to hypoxia, in order to ensure that the compound can reach its targeted proteins inside the cells before starting hypoxic response.
If your test compound is small molecule, there should be no difference between immediately subject to hypoxia and 30 min incubation before hypoxia.
But if your test compound is large molecule or nanoparticle, it should be incubated for a period to allow the test compound is uptaken by endocytosis and released into cytoplasm before starting hypoxic response.
In another paper, I use a different treatment schedule. See in "Curcumin resistance induced by hypoxia in HepG2 cells is mediated by multidrug-resistance-associated proteins" Anticancer Res. 2012 Dec;32(12):5337-42. The cells were pre-incubated under hypoxia for 24 h, then bring it out for drug treatment and immediately return it to hypoxia for further 24 h incubation. For some compounds, the pre-incubation under hypoxia for 24 h before treatment gives results different from treatment before starting hypoxia. Because pre-hypoxia can up-regulate drug transporter genes that can pump-out the compound. This pre-hypoxia model mimics the situation that patients already have hypoxic tumors before drug treatment.
Good luck ;)
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