Hello Beth Crofts,

1. Do I add 1ml of complete media to one of the wells for the control at this stage or do I wait unit I am treating the plates to do that?

You seed the cells in each well in such a way that they become 80% confluent after 24 hours. Perform a pilot experiment to optimize the seeding density and choose that cell concentration which may become 80% confluent after 24 hours before you start your MTT assay. You do not add 1ml of complete media to one of the wells as control at the time of seeding the cells in the plate. Control and test wells have to be treated equally. So, you should wait until you treat the cells with the drug.

2. Also what order do I do things? So I seed the cells on days 1. Day 2 I treat them. Day 3 do I transfer the drug free plates to new plates? and then at what stage do I do the MTT part of it?

You seed the cells on Day 1. Treat the cells with the drug on Day 2. Add 1ml media instead of the drug to the control well on Day 2. You may treat the cells for 2 or 3 time points namely 24hours, 48 hours and 72 hours. So, you will have to plate three 6-well plates for three time points. Perform MTT assay after 24 hours for the plate labelled as 24 hours, perform MTT assay after 48 hours for plate labelled as 48 hours and so on. Calculate the IC50 value for each time point.

While performing MTT assay after 24 hours, aspirate supernatant (media containing the drug) after 24 hours from the wells of the plate labelled as 24 hours. Add 1 ml of fresh culture medium which would include the MTT reagent (0.5 mg/ml) to each well and incubate for 1-4 additional hours at 37 degree C. The time of incubation with MTT reagent has to be optimized so that sufficient signal is generated. Formation of formazan crystals can be checked by observing the cells under inverted microscope periodically during incubation. The crystals appear as needle-shaped and dark purple coloured intracellular precipitates. Longer incubation period may be required if adequate amount of crystals are not formed. Subsequently, 1ml of solubilization solution may be added into each well and the plate incubated overnight at 37 degree C.

The solubilized formazan product should be diluted with PBS (it would be better to measure an original concentration before dilution. Then you may decide how many times you need to dilute the solubilized formazan product). Later transfer 100ul of the solubilized formazan product to a 96 well-plate to measure the absorbance at 550 nm with a reference of 650 nm. Reference wavelength is required for non-specific readings.

Similarly, perform the MTT assay for the plates labelled as 48 hours and 72 hours after 48 hours and 72 hours respectively, as mentioned above.

Best.