In MTT or Alamar Blu test for cytotoxicity assay by using UV spectrophotometery, I saw many biologist researchers that use absorbance even more than 1 for their calculations. Is it correct? As I am chemist, from the Beer–Lambert law, we Learned the absorbance more than 0.9 and lower than 0.15 is not linearly have a relation with concentration. Hence, they should dilute all samples? or doing something else?
Some colorimetric assays as mtt, cell titer blue or Crystal violet ( i dont know the alcian blue) can provide very high assorbances.
The best way when you obtain a value >1 is dilute the sample. measure again and calcolate the True value considering the diluction factor. Of course you have to dilute also the blank (eh only mtt) and subctract sample values for it before the moltiplication.
in this way you will see as Some modern spectrophotometrs supply good linearity also up to 1.5-2 but i think that is it ever better perform solution and remain in the 0.1-1 range.
Of course if you have very high values in this way you solve the problem of the reader saturation but you risk to have problem related to the colorimetric agent or substrate amount that can become a limiting factor, so before start is is better to run a preoliminary experiment to assess which is the maximun number of cells that you can
measure with the probe and then yuo can perform dilutions to cover absorbance values with-in this range the can change probe by probe and in function of the probe concentration that you use for the assay.
good luck
Manuele
Yes, that is correct, abs values up to 1.25 can be used. Many kit providers mention the abs range for untreated cells to be 0,75 - 1,25, including the one from ATCC.
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