I have been running drug treatment assays on cell lines in 96-well plates.
Following 72 hours incubation, I perform a MTT assay to assess cell viability following the treatment.
I am frequently seeing lower readings from cells at the center of the plate compared to those at the edges.
I have attached the Absorbance readings from four such experiments.
Columns 1 - 9 are Treatment wells.
Column 10 are Untreated wells
Column 11 are Blank wells
Any explanations for this or any advice on how to tackle the issue would be hugely appreciated!
Thanks!
These problems are nearly always related to evaporation of media.
Make sure the incubator has has a tray of water in the bottom
Also add sterile water in the spaces between wells as well.
Hi,
that sounds like a quite extreme edge effect, evaporation of medium in the outer wells during treatment may be the reason, resulting in higher real substance concentrations as already noted.
Beyond that MTT is very sensitive to pH shifts in medium right before solubilization of the dye when large amounts of residual medium is present, and this shift will be more prominent in medium in the perimeter of the plates outside of CO2 atmosphere. Make sure to remove as much of the MTT incubation medium from the wells as possible, maybe even try to wash the wells with PBS if you observe a good adherence to the wells.
What kind of solvent do you use before photometric measurement? DMSO with ammonia can alleviate this effect, see the following article for details:
Article Ammonia-containing dimethyl sulfoxide: An improved solvent f...
Did you get out the medium (medium + compound) in MTT assay?
maybe your compound can react with MTT and the salt didn't formed.
If I believe it has to do with edge effect...make sure media is not evaporated...or you may check if same compound used in extreme well and in centre gives different read...then it's definitely due to evaporated media
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