Nuclear factor-kappa B (NF-κB) β-lactamase and luciferase reporter assays were
performed as described previously.30 (link) NF-κB-bla cells or NF-kB-luc cells were dissociated with 0.05%
trypsin/EDTA, resuspended in assay medium, and dispensed at 2000 cells/5 μL/well
in a 1536-well black clear- or 2000 cells/4µL/well in a white solid-bottom plate
(Greiner Bio-One) using a BioRAPTR Flying Reagent Dispenser (FRD) (Beckman
Coulter, Pasadena, CA). Twenty-three nanoliters of compound was transferred to
the assay plate by a Wako Pintool station (Wako Automation). One microliter of
medium with or without 1 ng/mL TNF-α was dispensed by an FRD. After the plates
were incubated for 5 h at 37 °C, 1 μL of LiveBLAzer B/G FRET substrate (Thermo
Fisher) detection mixture and 5 μL of ONE-Glo luciferase assay reagent (Promega)
were added. The plates were incubated at RT for 2 h and 30 min, respectively,
and fluorescence intensity (405 nm excitation, 460 and 530 nm emissions) and
luminescence were measured by an Envision plate reader and a ViewLux plate
reader (PerkinElmer), respectively.
performed as described previously.30 (link) NF-κB-bla cells or NF-kB-luc cells were dissociated with 0.05%
trypsin/EDTA, resuspended in assay medium, and dispensed at 2000 cells/5 μL/well
in a 1536-well black clear- or 2000 cells/4µL/well in a white solid-bottom plate
(Greiner Bio-One) using a BioRAPTR Flying Reagent Dispenser (FRD) (Beckman
Coulter, Pasadena, CA). Twenty-three nanoliters of compound was transferred to
the assay plate by a Wako Pintool station (Wako Automation). One microliter of
medium with or without 1 ng/mL TNF-α was dispensed by an FRD. After the plates
were incubated for 5 h at 37 °C, 1 μL of LiveBLAzer B/G FRET substrate (Thermo
Fisher) detection mixture and 5 μL of ONE-Glo luciferase assay reagent (Promega)
were added. The plates were incubated at RT for 2 h and 30 min, respectively,
and fluorescence intensity (405 nm excitation, 460 and 530 nm emissions) and
luminescence were measured by an Envision plate reader and a ViewLux plate
reader (PerkinElmer), respectively.
