Fluostar omega 96 microplate reader

The two-step bioluminescent assay (MAO-Glo™ Assay Systems; Promega, Madison, WI, United States) was performed in Nunc white, 96-well, flat-bottom assay plates (Life Technologies Europe B.V., Zug, Switzerland) as described previously (Van Der Toorn et al., 2019 (link)). Fluostar Omega 96 Microplate reader (BMG LABTECH GmbH, Ortenberg, Germany) was used to measure the luminescent signal and to determine the IC₅₀ (half maximal inhibitory concentration). Z’ scores were between 0.75 and 0.86 for both MAO A and B assays. The constant (Km) of MAO A was 17.1 and 2.6 µM for MAO B. The substrate concentrations (S) were 20 µM for MAO A assay and 3 µM for MAO B. The Ki values referred in the discussion were calculated accordingly to the following formula: $Ki=I{C}_{50}/\left(1+\frac{Km}{S}\right)$ .

The two-step bioluminescent assay was performed in Nunc white, 96-well, flat-bottom assay plates (Life Technologies Europe B.V., Zug, Switzerland). In the MAO reaction, recombinant MAO (MAO-A and MAO-B, 0.4 and 0.1 U/well of microsomal protein, respectively) was incubated with derivative of beetle luciferin ((4S)-4,5-dihydro-2-(6-hydroxybenzothiazolyl)-4-thiazolecarboxylic acid) substrate and sample/vehicle for 1 h at room temperature in a 50-μL reaction mixture. The beetle luciferin substrate concentrations were varied for determining the kinetic constants, but all subsequent experiments used the substrate at the Michaelis constant (Km) values of 20 μM and 3 μM (MAO-A and MAO-B, respectively). In the luciferin detection reaction, 50 μL of luciferin detection reagent (Promega) was added to the MAO reaction. After a 1-h incubation period, the luminescent signal was measured by using a Fluostar Omega 96 Microplate reader (BMG LABTECH GmbH, Ortenberg, Germany).