Hello everyone,
I have one question regarding the MTT assay, as the picture shows, why do some cells take MTT color but some do not? Cells in some wells even only take the edge of color. All the cells are intact under microscope.
Does anyone have such experience to solve this problem?
Thanks for your time.
Pengyan
Would be more clear if you could also share the experiment design and conditions. Like your cell type, seeding amount, treatment. Without these details, there are something I would do:
The first thing I would check is if the cells are alive. You can stain them with trypan blue. Once the cells are dead they could be stained. Then it means they won't uptake or metabolite the MTT. Also, if the cells are at senescence phase, they will also metabolite MTT. So, check if there are any morphological change could be considered.
P.S. seems controls are not quite right.
Hello Pengyan fa
For cells in the log phase growth, the amount of formazan product is generally proportional to the number of metabolically active viable cells. Culture conditions that alter the metabolism of the cells will likely affect the rate of MTT reduction into formazan. For instance, when adherent cells in culture approach confluence and growth becomes contact inhibited, metabolism may slow down and the amount MTT reduction per cell will be lower. This type of situation will lead to a loss of linearity between absorbance and cell number.
Other adverse culture conditions such as altered pH or depletion of essential nutrients such as glucose may lead to a change in the ability of cells to reduce MTT.
You may need to optimize your seeding density to achieve the desired level of confluency within a convenient period so that at the end of the incubation period, the control (consisting of cells only) should be about 80% confluent. The cytotoxic effect of the drug candidate may be influenced by cell density. Generally, the greater is the confluency, lower will be the toxicity. Also, avoid variation in seeding density during plating of cells which could occur at times when certain cell lines tend to form clumps rather than a suspension of individual cells.
Hope this helps.
Best.
Pengyan fa hello;
In the MTT protocol, water-insoluble formazon crystals are formed. Therefore, these crystals need to be dissolved, which is only possible with DMSO. My advice to you is to review the protocol of the kit you use and repeat the experiment.
Good work.
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