This MLR will be done using two different strains of mice that is why it is necessary to inactivate the T-cells, so that possible proliferation can be observed.
Normally tha final concentration is in the range of 10 to 50 ug/ml of mitomycin C in culture media.
Incubation times could also vary between 0,5 hours to 3 (being 2 hours the most used) at 37º in an humidified incubator 5% CO2.
then you have to remove carefully the supernatant (even you can do a centrigue before) to take off all the mitomicyn, and then a couple of was either with fresh culture media or PBS, depending on what you want to do afterwards.
If you are not sure of incubation times and mitomycin doses, you should set a proper experiment making different times and dose, and the stimulating cells with PHA and measuring stimulation.