Dear all, for the case of MTT assay, does the volume of formazan crystal solubilising agent such as DMSO affect the optical density (abs) of the well?
Yes of course.
when you use more abs will get diluted and gives less reading. better to keep constant volume in each well
Yes, for 2 reasons, concentration and path length. The absorption of light or optical density depends on both concentration and path length of the light through the well. It is important to know what volume is needed in a well by your spectrophotometer, and always use that volume because this is the path length that the instrument uses to calculate your Absorbance. If the path length is wrong, then so is the concentration determined from the absorbance. And obviously, if you use more volume, then the concentration decreases, which also decreases the Absorbance and can lead to increased errors as the readings become too low.
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