Hi everyone.
I'm using MTT for cytotoxicity experiments with Vero cells. My question is about the calculations. I measured my samples at two wavelengths:
- 570 nm
- Reference: 630 nm
Please let me know if this is how we should do the calculations:
Should I correct each of my OD570 by subtracting the mean blank at 570 (Sample570 - MeanBlank570)
And correct each of my OD 630 by subtracting the mean blank at 630 (Sample 630 - MeanBlank630)
And then finally Adjust as follows: (Corrected570 - Corrected 630)
Is this how we use both Blank and Reference Wavelength in MTT calculations?
Hello Sueden de Souza,
It is much more easy to use one wavelength. I did a cytotoxicity assay on Monkey kidney epithelial cells which are cells from which Vero cells were isolated. Use a single wavelength (570nm), use the OD of control (well that has cells and vehicle but void of the compound under investigation for cytotoxicity). Since you have done serial dilutions inorder to calculate the CC50, substrate OD of concentration 1 from OD of control and divide what you have by the OD of control. Then multiply your answer by 100% to have % inhibition of Vero cells (do this for all dilutions). You will have % inhibition for all dilutions depending on the number of serial dilutions you did for a single compound or extract or ....... Use these values to calculate the CC50 with the help of a statistical software.
Check the article attached below for more information
Hello Sueden de Souza
I would suggest the following:
The reference wavelength is used to correct for optical variation. It allows individual corrections to be made for each well due to variation in the well architecture (for instance, well-to-well variation, control well and treatment well variation, etc) caused due to many reasons such as production defect or due to scratch, which in turn could change the apparent background absorbance value of each well. So, you will have to subtract background absorbance from signal absorbance (OD570 - OD630) to obtain normalized absorbance values for each well including the blank.
After having normalized the values for each well (including the blank) for the reference wavelength, you may calculate the mean value for the blank (blank which has been taken in triplicate) and for the sample value (sample which has been taken in triplicate) at 570nm, after having removed the outliers.
Then finally adjust as follows: (Sample (Mean value) at 570nm – Blank (Mean value) at 570nm). This will give you the final value for your sample in MTT assay.
Best.
Thank you very much for all your answers!! @Malcolm Nobre, that's very helpful.
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