Hello,

I am conducting an evaluation of the effect of a peptide on the C6/36 cell line using the resazurin reduction method. I have cultured the cells in L15 medium, supplemented with 20% fetal bovine serum and 20 mM HEPES. The cells were plated at a density of 40,000 cells per well in a 96-well plate and incubated at 28 °C in the absence of CO2 for 24 hours.

After that, I replaced the medium with fresh medium containing the peptide at different concentrations and incubated them again for 24 hours under the same conditions. Subsequently, I added 10 µL of Alamar Blue to each well and measured the reduction of resazurin to resorufin after 5 hours of incubation at 28 °C, using a microplate reader (Varioskan) at wavelengths of 560 and 590 nm.

However, I do not observe any metabolism of resazurin to resorufin, as there is no visible change and the pink, fluorescent compound is not formed. What factors could be affecting this result?