Hi everyone, I'm having some trouble with classical MTT assay. when taking OD at 570nm I'm getting invalid values like 6.000. what causes more formazan crystal formation in the well than the cells could convert from MTT, is it due to some extracellular reaction?
Hello Tomcy Tomy Anjilikal
Which cells have you used for the MTT assay?
Any suitable reducing agent will produce a 570 nm absorbing product, whether it be an electron carrier in the mitochondrion or a simple chemical reducing agent at a moderately high concentration in the culture media e.g., some thiols, ascorbate etc.
Hello Tomcy Tomy Anjilikal
It is known that sperm with rich mitochondria is a highly active cell and can quickly convert MTT, which shows that the reduction of MTT by spermatozoa could be faster than other cell types. The incubation time for MTT in MTT assay is from a minimum of 30 mins to a maximum of 4 hours depending on the cell type and culture conditions.
Since you are dealing with a cell type that is highly active, I would suggest you determine the optimal time for MTT reduction for spermatogonial stem cells. An incubation time with MTT solution for not more than 1 hour should suffice and then solubilize with DMSO. But you need to optimize the incubation time yourself.
Best.
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