Spectramax m5 fluorescence microplate reader

For in vitro characterization of recombinant luciferases, luminescence intensity was measured in white opaque 384-well microplates (OptiPlate-384 HS, PerkinElmer Inc.) using 2104 EnVision Multilabel Plate Reader (PerkinElmer Inc.). The assay reagent contained 50 mM Tris-HCl, pH 8.0, 150 mM NaCl and 50 μM Nano-Glo substrate (furimazine, FZ; Promega Corporation) or 100 μM coelenterazine; CLZ (Biosynth International, Inc.). Luminescence intensity was recorded 3.0 min after adding the assay reagent to the respective luciferase dilutions (5×10⁻² – 1.56×10⁻³ μM). Emission spectral scans of all recombinant luciferases (50 nM each) were performed in white opaque 96-well microplates (OptiPlate-96, PerkinElmer Inc.) using SpectraMax M5 fluorescence microplate reader (Molecular Devices, LLC). Emission spectra were recorded from 390 nm to 600 nm using the integration time of 1000 ms with 5-nm step increments. The assay reagent for the emission spectral scans contained 50 mM Tris-HCl, pH 8.0, 150 mM NaCl and 50 μM FZ) (Promega Corporation) or 100 μM CLZ (Biosynth International, Inc.).