MAO activity was measured fluorometrically in permeabilized cells in an assay using
10-acetyl-3,7-dihyrdoxyphenoxazine (Amplex Red reagent, Life Technologies), in
combination with horseradish peroxidase (HRP), to detect
H2O2 generated during substrate catabolism. Following
exposure to H2O2, recombinant MAO-A and –B (Sigma)
were spun down and resuspended in PBS. MAO protein (3.5 μg/reaction) was
diluted in PBS containing 10 μM Amplex Red and 7.5 μg/ml HRP and
added into black opaque 96-well microplates. Amplex Red fluorescence was
measured kinetically with a fluorescence microplate reader (Fluoroskan Ascent
Microplate fluorometer, Thermo Fisher Scientific, excitation/emission
wavelengths 560/590 nm), at baseline and following addition of substrates. MAO-A
and B activities were assayed using tyramine and phenylethylamine as substrates,
respectively. The velocity of H2O2 production was
calculated from a calibration curve obtained adding known amounts of
H2O2 and results were expressed as rate of
H2O2 formation per μg protein.
10-acetyl-3,7-dihyrdoxyphenoxazine (Amplex Red reagent, Life Technologies), in
combination with horseradish peroxidase (HRP), to detect
H2O2 generated during substrate catabolism. Following
exposure to H2O2, recombinant MAO-A and –B (Sigma)
were spun down and resuspended in PBS. MAO protein (3.5 μg/reaction) was
diluted in PBS containing 10 μM Amplex Red and 7.5 μg/ml HRP and
added into black opaque 96-well microplates. Amplex Red fluorescence was
measured kinetically with a fluorescence microplate reader (Fluoroskan Ascent
Microplate fluorometer, Thermo Fisher Scientific, excitation/emission
wavelengths 560/590 nm), at baseline and following addition of substrates. MAO-A
and B activities were assayed using tyramine and phenylethylamine as substrates,
respectively. The velocity of H2O2 production was
calculated from a calibration curve obtained adding known amounts of
H2O2 and results were expressed as rate of
H2O2 formation per μg protein.