Optimization of seeding cells number for MTT assay - (Jan/21/2008 )

Hi, I plan to seed different cells number into a 96-well plate for optimization of MTT assay. What is the suitable range of cells number I should load? 1000-10,000 cells per well? The cell line used here is adherent mammalian cell line. Then I need to calculate the volume needed for all different cells number and load into the well one by one?

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You should keep final volume of medium the same. The cell number is cell type dependent. Less cell number typically have more sensitivity toward treatment (cell death, for example). Sometimes, you just have to test it out experimentally. No one can answer/predict everything (the outcome) for you.

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Thanks for your reply. I will try an error and relate the cell numbers to the optical density obtained.

What is the optimum optical density range should be choosen when determine the optimum seeding cells number? between 0.8-1.2? The max OD value that can be detected by the ELISA Plate Reader is 4.0.

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I used XTT before... it is a similar assay but the end product is soluble compare to MTT...

If i'm not mistaken, in the information sheet of the kit, there mentioned the minimal and maximum numbers to use.
However, i did optimise by plating different numbers of cells (eg: 1000/cm2, 10000/cm2, 100000/cm2..... etc) in 96 well plate and see at which cell density, it will give a consistent reading.

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So, we have to choose the cell density which gives the consistent reading and within the detactable limit right?
Thanks.

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of course...

lets say the limit of detection is 1000. but you plate 10,000....
after treatment, only 50% of cells viable = 5000 viable cells.
but your MTT couldn't detect it and still give you the same colour like your control... then you won't know you actually already have half of your cells died.

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