Hello, everybody,
I am performing an experiment with the MG-63 cell line in 3 concentrations: 70,000 cells/well, 60,000 cells/well and 50,000 cells/well, and I want to compare the mtt and mts assay because I will finally make an mts. I use 48-well plates with 1 ml of medium in each well and I don't know what amount of mtt reagent to use. I know that for 0.5 ml we add 50 microliters of the first reagent and 300 microliters of the second.
So, how much mtt reagent should I add? and in the case of the mts assay, how long should I wait to read the plate?
thank you
Starting from your calculation, you have to double the amounts when using the doubled volume, compared to experiments done earlier.
However, to perform a correct calculation you need the corresponding and correct concentrations of MTT etc. in your solution/experiment/setting.
You need to have 5 mg/ml MTT in your well to proper MTT concentration. Good luck!
Estefanía López
In 48 well plates, MTT as 25μL of 0.5mg/ml stock solution were dispensed to the treated wells and incubated at 37°C for 4 hours. Thereafter, the treatment medium was gently removed from the wells, cells were washed using PBS and 50μL of Dimethyl sulfoxide were added to each well to dissolve the purple formazan crystals.
The absorbance was conducted at 570nm using the Dynatech MR5000 spectrophotometer. The absorbance values at 570nm were relative to the number of residual viable cells. Results were determined as the mean of three independent experiments. The viability percentage was calculated as following:
Viability percentage = X Absorbance of test dilution ×100
X Absorbance mean of control
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