You are highly advised to do a cell number titration. This is because you cell growth rate and the number of days will determine the number of cells.
Basically, your untreated cells need to be growing exponentially and still be in this phase when you "stop" the assay by adding MTT. If your control cells start slowing down because of medium or plate limitations, your MTT reading will be false. If in fact they are at a non-growing phase, then your treated cells which are going to be a much lower density can have a higher metabolic activity (which MTT determines) and so appear to be less "inhibited" with respect to control cells.
Thus, if you do an assay for 3 days, you should have a cell number that can double the MTT absorbance if they were incubated for another day.
Finally, the upper limit of absorbance value for any reader >1.2 - should not be exceeded if you can avoid it,. Thus, high cell number can cause absorbance saturation and lower estimation of total cells.
Good luck !
You are highly advised to do a cell number titration. This is because you cell growth rate and the number of days will determine the number of cells.
Basically, your untreated cells need to be growing exponentially and still be in this phase when you "stop" the assay by adding MTT. If your control cells start slowing down because of medium or plate limitations, your MTT reading will be false. If in fact they are at a non-growing phase, then your treated cells which are going to be a much lower density can have a higher metabolic activity (which MTT determines) and so appear to be less "inhibited" with respect to control cells.
Thus, if you do an assay for 3 days, you should have a cell number that can double the MTT absorbance if they were incubated for another day.
Finally, the upper limit of absorbance value for any reader >1.2 - should not be exceeded if you can avoid it,. Thus, high cell number can cause absorbance saturation and lower estimation of total cells.
Good luck !
As mentioned above, it depends on the cell line you're using, their growth rate, what experiment you're doing and what you're trying to test. A titration of cell numbers between 1,000 & 10,000 per well would be a good place to start.
its depend on cell type and its growth rate; and also on number of days of your experiment. For fast growth rate cultures, 1000 cells/well in 96 well plate/72hr will be work out.
To determine animal cells number to seed in 96 well plate for MTT, you better do titration to cell number. First, seed 100,000 cells per well per 200 ul into 1st roll of 96 well plate and make 2 fold dilution for remaining 7 rolls. Remaining 7 rolls to be filled with 100 ul media first. Then do two fold dilution. By doing so, 2nd roll will become 50,000 cells per well per 100 ul, 3rd roll becomes 250,000 so on and so for for the rest. Do like this 3 plates, if you are thinking to do MTT for 3 days (seeding day 0, treatment day 1 and reading day 2; all 24 hrs interval).
The first plate is to read OD value at 3 hrs after seeding. This will give you OD of seeding day. Take out 1st plate and add MTT and incubate for 3 hrs. Read OD value of each respective cells number after 3 hrs of seeding.
Next at 1st day, take out another plate and do the same. Record the OD value.
At 2nd day, take out another plate and do the same to record the OD.
From those above 3 plates reading, Day 0 to Day 2 (total 3 days), Check which maximum cell number still fall in exponential phase from day 0 till day 2. Choose this maximum number of cells especially in cancer cells. Cancer cells are highly packed tumor mass in real situation. This is why we have to choose max cells number that still give you their growth in exponential phase throughout seeding day at day 0, treating day at day 1 and reading day at day 2.
For normal cells, it is also suggested to get max cells number too because cells in real situation is highly packed and not like mono-layer cells in 96 well plate. This is why it is highly important to mimic the real situations so that your MTT result is more applicable to real life situation.
If you seed very less cells e.g. 1000 to 5000 cells, yes they will definitely be in exponential phase within 3 days from seeding. However, your IC50 value is very low and it is not reflected to real life situation.
All in all, generally, I noticed that cells were seeded (animal cells), in average 20,000 to 25,000 cells per well per 100 ul in 96 well plate (for cells doubling time about 24hrs to 36 hrs). Unless your cells grow very fast or grow very slow, then seeding number will be varied. Hope this helps.
Dear Jamail Lu,
Thank you for the explanation its very informative.
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