i am wondering why i have to do colony formation assay after MTT assay. there are two opinions which i learned from those people did that experiment. the first opinion is they do this assay to confirm their MTT assay results through real pictures, not only numbers or cell viability percentage. the second opinion is they do this assay to confirm their results through a longer time period rather than MTT assay. now i confused which one is correct or may be both of the are wrong!!!
i will appreciate if anyone can save me!!
Seyed,
it all depends on what pathways/molecules and in what cell lines you're studying. You may see cytostatic effect or no effect in a short timeframe of MTT assay and would need to grow cells in colonies (2D or 3D) to observe cytotoxic effect.
Based on my experience in multiple cell lines and various viability assays in industrial screening, the major differential is a pathway that you're targeting.
Hi Seyed,
You don't have to do colony formation assay after MTT assay. You can confirm MTT data by trypan blue exclusion assay, or other viability-measuring assays. MTT assay and colony formation assay are two different assays, which implies the presence of a lot of variables. Hence, the first opinion does not apply (to confirm their MTT assay results). Regarding the second opinion, I would agree but to a certain extent. Colony formation assay is considered a 3D cell culture assay where cells grow independently of a substrate (also known as anchorage-independent growth). Only cancer cells can grow without a substrate, normal cells fail to do so. Hence, this assay was primarily used as a transformation assay. It can be also used in a 3D context, particularly when studying long-term effects of drugs on anchorage-independent growth of cancer cells.
Best,
Patrick
Each method evaluates different aspect of cellular responses. MTT, MTS or assays of this sort analyze cellular metabolism. Colony formation assay analyzes the cellular clonogenic potential. Viability can be measured with a dye exclusion assay, e.g., using trypan blue. For example, prematurely senescent cells are clonogenically inactive (do not form colonies), but are metabolically active and viable. So, the choice of the method depends on what you wish to look at.
You don't need to perform both assay. You can chose according to your test ojective. We have tested many chemicals using both assay so far. Results obtained in both assays show similar tendency. However, the CF(colony formation) assay respond more lower dose of chemicals than MTT assay. The reason is that, in the CF assay, there is no cell-cell comunication to protect chemical hazardous because cells numbers plated are about 100 cells in each 60mm dish, and also cells are exposed long period, almost one week, than MTT assay.
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