If the compound's color is the only problem, then you can use the compound-alone control to measure the color and subtract that value from the cytotoxicity assay at each compound concentration in order to correct for the color.

However, if the compound is reacting chemically with something in the assay, then you should think carefully about whether that compound should be used, since its cytotoxicity may be non-specific. You could also consider using a different method to measure cytotoxicity that does not suffer from interference by the compound.

@Adam

Thank you, I have done with compound alone control, unfortunately, the treatment is also giving a lower value than LDH assay control and even water control.

What kind of other assays would help? Using MTT again would be a problem due to the colors

Here is a list of methods.

https://www.abcam.com/kits/cytotoxicity-assays

change the media

pH may be playing a mjr role so try to control

Sometimes presence of salts also lead to such kind

You can try MTT or resazurin test (similar to MTT only faster and rather cheaper). However, before adding the MTT reagent, you should replace the DMEM containing the test compound with pure DMEM (without serum) and only then add the MTT (or resasurin). Good luck.

Rajagopal Kammara and Iwona Zwolak

Thank you for your answers. Yeah, I agree about the pH change.

Even with MTT or resazurin, it will be the same problem because both are measured with color intensity. I would perform a trypan blue exclusion assay instead of any of these absorbance assays and see.

There are a few possible solutions to your problem: