We have irradiated A549 cells in 96 well-plate with 2Gy, 4 Gy 8Gy and 16 Gy doses and after 5 or 7 days the viability of cells decreased only to the 60 % with XTT test. It means these radiation doses are not killing our cells. Why? Any researcher have the same experiences?
I recommend you to search on the literature what is the behaviour of these cells towards radiation. Some cells, (ex: fibroblasts) are somehow resistants to radiation, mostly undergoing senescence when exposed to IR. On the other hand, other cells (ex: linfocites) are very susceptible to IR.
I suggest you to read this article, http://www.ncbi.nlm.nih.gov/pubmed/12563311
Another issue that you have to consider is what is the ratio of the irradiation (Gy/min). If the ratio is low (for example if you're using X-rays), maybe you are giving the cell too much time to repair the damage.
Good luck!
I checked and it seems that lung cells are indeed resistants to IR. I'm not sure about A549, but it's possible that it is too
thank you for your suggestions..I had not seen the resistance of A549 cells but it is possible and explain this situations. I think I should change the cell line..
Hi Nilufer even though I will agree with Rajao about some cells being more resistant to RI than others, do you treat the cells with different concentrations of your agent and just incubate them for 5 to 7 days and check their viabilities? if so it could also be that the cells are recovering from the RI effect. I will suggest that you do a time dependent study and see if there are any changes in the viabilities. What I mean is, after treating your cells, you could incubate them for say 2, 4, 6, 12, 16, 24, 42, 72 hrs and so on up to the seven days and see if the viabilities are similar before concluding.
Depending on the cell line we use different viability assays since differences in results are sometimes surprisingly big. For epithelial cancer cells I favour the good old crystal violet stain. It is by the way also the cheapest. Additionally, you have a chance to get a visual impression before dissolving the bound CV.
Concerning the amount of irradiation you are using: it will most likely be too low to kill A549 to a higher extent. Other people use also colony formation or similar to analyze influence of irradiation.
Michael thank you for your suggestions. we can not use colony formation assay because we purchased a 40 nm nanogold solutions at very low density and it is not suitable for colony formation test. Cheu Manka these are very good suggestions and we try to irradiate for 2, 4, 6, 12, 16, 24, 42, 72 hrs and so on up to the seven days..
we use nanogold to investigate the radyosensitive effect of it on A549 cells..
Hi
You probably need to increase the dose to 30Gy-50Gy from a gamma source. Mouse fibroblasts usually need 30Gy for growth arrest and human skin fibroblasts up to 50Gy. The range you have been using will probably be inducing apoptosis rather than arrest
hope this helps
kay
thank you for all suggestions. we have found finally a significant decrease to the 70% levels at 6 Gy dose with nanogold..
You could already treat your cells at high density and do a colony formation after to see the recovery of your cells. Irradiation can also induce senescence and autophagy! Apoptosis is not the only answer.....
Agree with most of the comments below - it is a good idea to compare results form different assays since they measure different things: (i.e. tetrazolium (MTT,WST, etc) measure metabolic hanges while dye exclusion measures membrane damage. The two may not be related. The standard viability assay for radiation exposure is clonogenic (colon based) assays since these are true proliferation assays. These do work with a variety of different NPs if the proper controls are aslo performed. 16Gy seems like a very leathal dose for A549 cells...
Dear Cordeiro your study was useful for us and gave the answers our questions. thank you..
Hi Nilufer
I came across this forum while i googled gamma irradiation of A549 cells.
I am irradiating A549 cells at 0, 10, 15, 20, and 30Gy. My MTT assay was no different from the control indicating that the cells were not facing any immeditae cytotoxic effect after irradiation.
I was wondering can a mammallian cells withstand such a high dose. I was doubting the Irradiator. Now i am confident that i am observing an expected phenomenon.
How measure different Gy in only one 96-well plate? or you measure it in seperatelly plates? You measure the viability palates by plates? or measure in one plate? Thank you
How measure different Gy in only one 96-well plate? or you measure it in seperatelly plates? You measure the viability palates by plates? or measure in one plate? Thank you
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