While performing the cell cytotoxicity test, my detection wavelength is 450 nm while the reference wavelength is 650 nm. Suppose I measure my cells viability (blank, control and test) with these parameters, shall I subtract/adjust the absorbance of blank at 450 nm or 650 nm during the final calculation?
I am doing WST-8 (its CCK-8 kit from SIgma) which measures formazan at 450 nm.
the reason of my doubt is:
All the samples are measured at 450 including blank and the reference wavelength is 650 nm. Shall I be subtracting the blank at 450 nm from samples at 450 or the blank at 650 from the samples at 450? and Why?
Thanks and Regards
Jiten
There are some good explanations here: https://www.researchgate.net/post/Why_in_MTT_assay_do_we_measure_the_optical_density_OD_at_two_different_wavelengths1
If you are doing MTT assays you should measure at 570nm and also at 630nm as reference value (background).After you should correct your values with the reference value.
Hi everyone,
I am not doing MTT.
I am doing WST-8 (its CCK-8 kit from SIgma) which measures formazan at 450 nm.
the reason of my doubt is:
All the samples are measured at 450 including blank and the reference wavelength is 650 nm. Shall I be subtracting the blank at 450 nm from samples at 450 or the blank at 650 from the samples at 450? and Why?
Hi Everyone, Thanks for your answers.
So my conclusion is:
From all the result at 450 nm
(I) First, Adjust the blank at 450 , and then
(ii) Adjust the reference at 650.
Any suggestion is more than welcome.
Regards
Jiten
Hi! Could anyone explain how to correct the measurement using the reference wavelength? Do I make an average of the measurements obtained with the reference wavelength for each group and subtract that average from the 450 nm measurement of each well? Or do I subtract the 630 nm value (not average) from the respective 450 nm value?
Sorry if my text is confusing. But when people say "adjust" using the reference, I'd like to know how exactly I should do that adjustment...
Thank you in advance!! :)
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