Nano glo luciferase assay buffer and substrate

Tg(gip:Nluc) fish were crossed with wild-type
fish to assess the effects of the compounds. Three 3 dpf zebrafish
larvae per well in 96-well plates were incubated in 200 μL E3 +
PTU medium containing either 1% DMSO or 20 μM chemicals from the
library (MedChemExpress), which was dissolved in DMSO. After three days
of treatment, 50 μL mixture of Nano-Glo™ Luciferase Assay
Buffer and Substrate (Promega) was added to each well and incubated for
2 hours at room temperature. By the end of incubation, the luminescence
signals were detected by a Glomax-96 microplate luminometer (Promega).
For each clutch, at least 8 wells were treated with DMSO and their
average value of luminescence were used to normalize the luminescence
values from other wells belonging to the same clutch. The effect of the
compounds was assessed by the calculation of Z-scores:
Z-score=(Luminescence value (compound-treated wells)-Average
Luminescence value (DMSO-treated wells)) / Standard Deviation
(DMSO-treated wells). Compounds with a Z-score > 2 were
considered to be positive and selected as primary hits. All the primary
hits were retested in an additional 4 wells to enable the selection of
secondary hits. The final hits were selected after testing potential
secondary hits for up to 10 wells. Both the average Z-score and the
frequency of positive results for each hit were used for determining the
final hits.