I evaluated the cytotoxicity of a compoud in WT cancer cells and drug-resistance cancer cell using MTT assay and colony formation assay. The cytotoxicity in two cells was significant different by using MTT assay. The compound showed weak effect on drug resistance cancer cell. However, the results of colony formation assay was differ from MTT assay. Low dose could inhibite the proliferation of both two cells. How to evaluate the cytotoxicity of this compound in the drug resistence cancer cell？Thanks.
MTT assays are many times (if not all the times) sold as "cytotoxic" assays. Even though they can indirectly measure cytotoxicity, these kind of assays measure the metabolic activity of the cells. Considering that living cells would be metabolically active and death cells won't, this assay is able to indirectly measure living cells vs death cells (cytotoxicity).
If you want to measure cytotoxicity and you have already seen a difference on the drug response depending on the assay that you use. It would be better to use an assay that measures death either by measuring markers of cell death like caspase 9 (apoptosis) or by measuring population of cells that are dead (early/late apoptosis, necrosis) or alive using flow cytometry and propidium iodide + AnnexinV. There are kits available for detecting cell death using the two approaches that i told you.
By the way... How did you measure cytotoxicity in the colony formation assay?
Yu Pan
Considering the information provided the difference that you get might be related to procedural differences.
In MTT assays, if i remember correctly, cells are seeded, treated, incubated and then read on a microplate reader. There are no "wash" steps. This implies that the response obtained comes from a bulk of attached and detached cells.
In the other hand, in the colony formation assays there are wash steps to remove unattached cells, leaving only attached cells for the analysis.
This could suggest that the differences that you are observing might be related to the unattached cells rather than the attached ones.
I know this seems very unlikely but the results that you are obtaining are equally uncommon.
Additionally, other things that might be involved in these contradictory results are the "attaching capacity" of the cell line used, the action mechanism of your drug, drug dynamics (confluence, drug availability like Herald stated), interpretation of the results, etc.
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