I followed the below protocol. Can anyone suggest me what seems like missing in my experimental? All the wells gave me the very close numbers (drug treated groups were the same with non-treated groups)
MCF-7 cells were seeded in a 96-well plate at density of 15× 10e3 cells/well and allowed to grow overnight
Recombinant human IFN-γ was then added to each well with a final concentration of 50 ng/mL. At the same time, various concentrations of drug were added to the cells.
After 48 h of incubation, 150 μL of the supernatants per well was transferred to a new 96-well plate.
Seventy-five microliter of 30% trichloroacetic acid was added into each well and the mixture was incubated at 50 °C for 30 min to hydrolyse N-formylkynurenine to kynurenine.
Next, supernatants were transferred to a new 96-well plate and mixed with equal volume of Ehrlich reagent (2% p-dimethylaminobenzaldehyde w/v in glacial acetic acid) and incubated for 15 min at RT.
Reaction product was measured at 490 nm by microplate reader.