I am doing MTT assays with two adherent cell lines. After a few adjustments, one cell line is all set up and the variation between technical repeats (3 wells) in a plate is minimal. But for the other cell line I'm still getting huge variations. I've adjusted the cell count, I'm not removing the whole media in a well to prevent cell death during treatment, and I have minimized treatment time. I'm also being very careful with the pipet not touching the cells while removing media. Do you have any suggestion on how to improve the results?
Dear Bahar Ksh, in our lab we use multichannel pipets to avoid the pipetting errors during experiments. We also mix very frequently the cells suspension / treatments / reagents before adding them. Have you tried this way?
Hi, I suggest optimizing the procedure to eliminate any medium change steps. The cell line used is crucial for minimizing well-to-well variation. For example, with HepG2 cells, carefully digest and pipette to ensure no cell aggregation before seeding. Additionally, shake the plates properly before incubation to ensure even cell distribution in all wells. Alternatively, consider changing from the MTT method to CTG or LDH.
If you observe your cells under a microscope, you will probably see that all the cells in a given well do not take and convert MTT in the same way, somme are heavily coloured and some other remain almost yellow suggesting a relatively strong heterogeneity from one cell to another even in the same well. What is the purpose of your test ? you should probably use another method to determine for example cell viability
Thank you Le Chang and Dominique Duval
I usually don't see any cell aggregation in wells under microscope, but I'll be more careful with shaking the plate after seeding. I totally agree with heterogeneity in cells' change of color and I believe this is the source of the issue. Dominique Duval do you know any non-fluorescent methods that can be used to check cell viability after drug treatment?
I guess that measurement of LDH (lactate dehydrogenase) release in the culture medium after treatment is a good non fluorescent method to determine cell death, but it is important to measure in control samples both LDH release in non treated cells and the total LDH content of cells after lysis of a non treated well. Trypan blue dye exclusion is time consuming but also gives relable results taking in account the viable and non viable cells present in the cell medium.
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