I am developing a cell viability assay where the decay in resorufin fluorescence (due to conversion to hyrdoresorufin) results in a narrow window in which to read out assay results. I am not concerned with maintaining a viable culture following viability analysis, and require a stable endpoint readout.
Is there an available protocol to circumvent the resorufin -> hydroresorufin conversion, or a method that would stall cellular respiration (preventing continued consumption of resazurin -> resorufin -> hydroresorufin) without affecting the resazurin/ resorufin fluorescence?
That seems to be a common issue without a straightforward answer. However, since evidence suggests that resazurin is converted into resorufin by NAD(P)H-dependent dehydrogenases, there are several things you could try to slow down or halt the enzymatic reactions. I would try either changing temperature (heat inactivation of enzymes or cooling the cells down to ice temp to slow the reaction), ionic detergent (1% SDS) to denature the enzymes, changing the pH of the medium to pH 5 or pH 9 to kill the cells, but this may slightly modify the wavelengths of resorufin excitation and emission. You could also try adding mitochondrial uncoupler (FCCP) and 2-deoxyglucose to increase mitochondrial NADH hydrolysis and slow cytoplasmic NADH synthesis to decrease NADH levels to slow the reaction. Another option may be just to add (pink) resorufin to the media instead of (blue) resazurin and monitor the the conversion of resorufin to (colorless) dihydroresorufin. But this reaction is reversible unlike the irreversible conversion of resazurin to resorufin.
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