Hello Ana María Bravo Valencia

MTT is converted to formazan by metabolically active cells. This formazan is initially insoluble and forms a precipitate. The issue arises when phenol red, a pH indicator in the medium, also precipitates, potentially interfering with absorbance readings. I suggest you use phenol red-free media for your experiments. Remove the culture medium before adding the solubilizing solution and measuring absorbance. Acidify the solubilizing solution (e.g., using SDS with HCl or acidified isopropanol). This changes the phenol red to a yellow color that has less overlap with the formazan absorption spectrum, minimizing interference.

The exact cell density should be optimized for your specific cell type. This can be done by testing a range of densities and determining which one provides the best signal-to-noise ratio.

Best,