Whatever the drug you use, you will always have an effect (if your compound is actually associated with growth inhibitory effects) when using cancer cells at 70-80% confluency and optical densitities between 0.8 and 1.2.

To determine these parameters using these cell lines in your "hands", you should plate the cell lines at 1,000 - 2,000 - 5,000 - 10,000 and 20,000 cells / 100 microL (thus per well) on a Monday and "read" the multiwells on Tuesday (day 1; D1), Wednesday (D2), D3, D4 , D7 (having thus a free week-end ...), D9 and D11.

You will thus determine the growth curve shape of each cell line and you will be able to determine the % of confluency or more precisely the number of cells / 100 µL well you must plate for having an OD of 0.8 - 1.2 three days after having added your compound.

Best regards

Robert

Whatever the drug you use, you will always have an effect (if your compound is actually associated with growth inhibitory effects) when using cancer cells at 70-80% confluency and optical densitities between 0.8 and 1.2.

To determine these parameters using these cell lines in your "hands", you should plate the cell lines at 1,000 - 2,000 - 5,000 - 10,000 and 20,000 cells / 100 microL (thus per well) on a Monday and "read" the multiwells on Tuesday (day 1; D1), Wednesday (D2), D3, D4 , D7 (having thus a free week-end ...), D9 and D11.

You will thus determine the growth curve shape of each cell line and you will be able to determine the % of confluency or more precisely the number of cells / 100 µL well you must plate for having an OD of 0.8 - 1.2 three days after having added your compound.

Best regards

Robert

you can use cell number between 5000 to 10,000 cells per 96 well culture plate and allowed to grow upto 80% confluency before treatment. In natural environment the cancer cells  grow as a clump/mass of cells that have a lots of cell to cell interaction and so many growth factors help in cell proliferation. To mimic cellular interaction in natural condition cell confluency should be at least 80% before treatment.

The cell confluence also depends on how long is your treatment. But whatever is the confluence (below 90%) the MTT should work if your treatment works at cell cycling and or viability. 

I agree with Robert, cells should be between 70-80%. You might have to wait 48  hours to see the effect of the chemotherapeutic agent (5-fluorouracil for example). But if there is a synergistic effect the treated cells will not be able to recover. Dose your cells with the drugs and plan to run the MTT 48 hours after dosing.

Good luck

I did mtt assay using both mcf and 231 cells. Per well mcf-7 should be 3000-4000 and 231 should be 1500-2000 in 100ul of medium

 I would like to add one query here about plate reading wave length. It is very confusing as some use 570 and ref 630, some other use 540 and ref 720, and also found 570 but no reference. So what is the ideal to apply for MTT assay plate reading? One literature mentioned that reference is not necessary but in his example, he uses reference too. 

I did growth curve too as suggested by others. However, I found that to get OD 0.75 to 1.2 after 3 days from seeding (1st day seeding, 2nd day treating, 3rd day reading), it is impossible to have 80-90% confluency at day 2 on which to treat the cells. If we have to wait for cells becoming 80 to 90% confluency, then at day 3, OD of non treat cells will never be between 0.75 to 1.2. 

So, how do you guys adjust those. 

Another thing that I noticed was that if we use 570 and ref 630, no matter how dark the color of the well, reader will give you max OD 1.5. Reader cannot distinguish darkness beyond the wavelength set unless you do not set Reference wave length. WIthout reference wave length you can get OD even fr 2.3 and over too. 

So my question here is setting reference wavelength is good or not? Why? If not necessary, why do all others always set reference at 630 and why not 720?