Hi everyone,
I am very new to MTT assay and I find myself stuck.
I plated 5,000 cells/well in a 96-well plate. Following recovery and incubation, I added 50uL MTT reagent to each well and incubated for 3 hours.
I did see formation of blue crystals in all wells. However, after removing the media and solubilizing with 0.04M HCl in isopropanol (200uL/well), I only see a yellow color-no purple.
Is there a reason why I would see yellow and not purple? Should I rinse the wells with PBS before solubilizing?
Thanks!
It may be that you don't have enough cells per well to create enough blue colour. With our MTT assay on spleenocytes we use 100,000 cells per well in a 96 well plate.
I might happen, if your cells are death or have very low activity.
I agree with all the above comments, another thing i would like to add is to be careful while removing the media.....
Seed 1x105 /200 uL, add 10uL MTT and then remove medium with MTT, add 200uL lysis buffer.
50uL MTT is too high, you need 10uL to cover area of well,
if used, 48 well, 20uL is enough.
good luck
hi Elizabeth Stewart,
As the others, I t might be your cells is not enough, death or low activity. But It is better before you treat with MTT check first your condition cells under microscope. you can put positive control too (e.g. untreated cells), so you can find the problem whether the amount of the cells, death cells or low activity. Actually you should put untreated cells to measure the activity of your sample. can you tell me about the concentration of your mtt?
hope help you, good luck
After solubilising the purple formazn crystals , it can turn out to yellow colour, purple coliur formation can be seen only aftr 3 hrs of incubation and if you hv seen the same colour of formazan crystals, it means your experimnt z working, you can put the control for the same to compare your results activity z lower, that you only see when you incubate your cells with MTT fr 3 hrs aftr this you are almost stopping your reaction.
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