cancer cells culture

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Patricia Artiles
Patricia Artiles's picture
cancer cells culture

Hello, My name is Patricia and I am working with cancer cells and MTT assay. I am getting some extracts from bacteria and cheking it at the MTT assay in order to get some extracts that kill cancer cells.
I have been in trouble when I tried to repeat the assay cheking the same extracts. I didn't get the same results. Does anybody kwon what is the MTT assay's efficiency? Maybe I am doing something wrong. Could anybody tell me something about that?

samm
samm's picture
Hi! There are three issues

Hi! There are three issues you need to consider:
1) the "window" for significant, reproduceable differences between control and treated populations(increase or decrease) should ideally be in excess of 3 fold. Differences of 1.5 fold or thereabouts may not show up because of the reasons below. Remember, MTT is a metabolic assay, and NOT a very sensitive indicator of proliferation/death - but it is easy, cheap and scaleable!
2) the base conditions of your cells - e.g. (A) cell density when assay was started, (B) plate confluency when cells were obtained for the assay, (C) time after plating/confluency when MTT was added, and (D) finally period of MTT treatment must be standardized initially.
3) the way the insoluble ppt is dissolved must be standardized for your cells - the acid alcohol with T-X100 o/n worked well for me.

Finally, always ensure you have untreated/vehicle treated cells in the same plate, and pulse them with MTT to serve as internal control for relative fold differences, as well as an appropriate medium alone blank. these controls must be present in every plate, if you have multiple plates. Do the assay in triplicates if possible, duplicates otherwise - do not trust single well conditions.