On day 1, cultured U2OS (PLC-EA and P23H-opsin-PK) cells were detached, resuspended and diluted to 3 × 10
5 cells/mL with assay medium. The cell suspension was dispensed at 20 μL/well into a 384-well, white-walled, clear-bottom, tissue culture-treated plate (Greiner Bio-one, Monroe, NC, USA) with a Multidrop dispenser (Thermo Scientific, Waltham, MA, USA). The 384-well plate was centrifuged at 300
g for 30 seconds, and then incubated at 37°C in 5% CO
2. On day 2 of incubation, a dosage series of 9-
cis-retinal working solution (5 times concentrated in the assay medium) was prepared. Dimethyl sulfoxide in the assay medium (at final concentration in the assay of 0.1%) was also provided as a negative control. Five microliters/well of either 9-
cis-retinal working solution or 0.5% DMSO was dispensed into each well of a 384-well plate containing the cell cultures. Each dose of 9-
cis-retinal was examined in triplicate, whereas the DMSO control was repeated 16 times. The plate was centrifuged at 300
g for 30 seconds and incubated at 37°C in 5% CO
2. On day 3, the reaction buffer was prepared as described in the instructions for the Gal-Screen System (Applied Biosystems), and dispensed into the prepared 384-well plate at 25 μL/well with the Multidrop dispenser. The plate was covered with foil and incubated at room temperature for 1 hour. The plate was read for luminescence with the EnVisionTM plate reader (PerkinElmer) with an integration time of 0.1 s/well. The averages and standard deviations of the relative luminescence unit (RLU) readouts from repeats of each condition are listed in
Supplementary Table S1, and plotted as the
y-values and error bars in the dose-response chart with the corresponding log
10[9-
cis-retinal] concentrations shown as the
x-values in
Figure 2B. The dose-response curve for 9-
cis-retinal was fitted by Origin8.1 software (OriginLab, Northampton, MA, USA), and the data range was selected to rule out data points at high dosages which reflected cell death with a subsequent decrease in luminescence. An observed EC
50 was obtained at the concentration of 9-
cis-retinal, which achieved 50% of the maximal experimental luminescence signal. The seeding density, and β-Gal reaction time were optimized by this protocol, ensuring a high
Z' value. Dimethyl sulfoxide was tested from 0.01% to 1%, which confirmed that addition of up to 0.5% DMSO did not affect the assay readout. The 100% value was measured at 5 μM 9-
cis-retinal, and the 0% value in 0.1% DMSO. Quality control (QC) of the assay was tested with the 100% and 0% values, each treated with 16 repeats in a 384-well plate. The QC parameters, the signal-to-background ratio (S/B ratio) and
Z' values were calculated as S/B ratio = Mean
100% control/Mean
0% control; and
Z' = 1–3 × (SD
0% control + SD
100% control)/|Mean
100% control − Mean
0% control|, with Mean as the average of each control readout.
28 In the QC experiment, an S/B ratio greater than 30 and
Z' greater than 0.70, suggested that the established protocol was reliable for scaling up the HTS (
Fig. 2B, inset).