Spectramax m5e plate reader

Selected hits were tested in 96-format white, flat-bottom plates with varied concentrations of an inhibitor (0.5-25 µM). Cells were plated at the density of 25,000 cell per well using a WellMate multichannel dispenser from Matrix (Thermo Fisher Scientific, Waltham, MA) and grown overnight on DMEM/F12+GlutaMAX (100 µl per well). Then an inhibitor was added, and the plates were incubated for a fixed time interval; the medium was removed, cells lysed, and luciferase activity was measured on a SpectraMax M5e platereader (Molecular Devices, Sunnyvale, CA) with BrightGlo™ reagent (Promega, Madison, WI). The reporter activation was normalized to the background luminescence. The effect of 0.5 mM N-acetylcysteine (NAC) was studied by simultaneous addition of 5 µL of 10 mM stock of NAC in water and 2 µL of 50x stock solutions of a flavonoid in DMSO.

The polyphenols were screened for DisA inhibition as previously described20 . Briefly, reactions containing 300 μ M ATP, 10 μ M coralyne, 3 mM KI, 20 μ M inhibitor and 1 μ M DisA in reaction buffer (40 mM Tris-HCl, pH 7.5, 100 mM NaCl and 10 mM MgCl₂) were set up in triplicates at 30 °C in 96 well plates. A Molecular Devices SpectraMax M5e plate reader was used to measure the fluorescence of coralyne with excitation and emission wavelengths of 420 nm and 475 nm respectively for 30 min with 2 min intervals.

Vero cells (ATCC) were transduced with a lentivirus expressing NanoLuc® luciferase (Vero-NLucP cells) as described previously⁹ . Vero-NLucP cells were plated (5000 cells/well) in a white clear-bottom 96-well plate and treated with toxin the following day. Following overnight incubation (20 h), luminescence signal was developed using the NanoGlo® Luciferase Assay kit (Promega) and read with a Spectramax M5e plate reader (Molecular Devices). Relative luminescence units (RLU) were normalized relative to untreated cells and data was analyzed with GraphPad Prism 7.0. For protein synthesis inhibition assays in HEK293T cells, cells were transiently transfected with the pNL3.2CMV (Promega) rather than by lentiviral transduction. Cells were plated in a 6-well plate at 1 × 10⁶ cells/well and transfected the following day with 3.0 µg of plasmid DNA, in a 3:1 ratio of FuGENE^®HD (Promega) transfection reagent to DNA. After 24 h, cells were re-plated at 5 × 10³ cells/well, in a white clear-bottom 96-well plate and treated the following day with toxin. After overnight incubation (20 h), luminescence signal was developed using the NanoGlo® Luciferase Assay kit (Promega) similar to above. Each construct was tested 3 times with 2 technical replicates (at a minimum). Wildtype and DPH4^-/- HEK293T cells were a gift from Dr. Mikko Taipale at the University of Toronto.

Experiments were performed as per the manufacturer’s instructions. Briefly, 100 nM of GTD enzyme was incubated in glucosylation buffer (see above) with various concentrations of inhibitor in a final volume of 16 μL. Reactions were started with the addition of 4 μL of UDP-glucose (50 μM final). Reactions were allowed to proceed at room temperature for 15 min. To stop the reaction, 10 μL were removed and added to a white, polystyrene 96-well half-area plate (Costar) containing 10 μL of UDP detection reagent. Plates were incubated at room temperature for 1 h, then luminescence was recorded on a SpectraMax M5e plate reader (Molecular Devices) with an integration time of 750 ms. Results were analyzed with SoftMax Pro 6.2.2 and GraphPad Prism 5.0.