Cell viability was measured using the RealTime-Glo™ MT Cell Viability Assay (Promega, Madison, WI, USA) while cell death was measured using the CellTox™ Green Cytotoxicity Assay (Promega). Both assays are nonlytic and homogeneous. In the former assay (glo, in the following text), the number of viable cells is proportional to the amount of luminescent signal, which is directly proportional to the amount of NanoLuc® substrate used in the NanoLuc® luciferase reaction. The NanoLuc® substrate is only produced in live cells where the cell-permeant prosubstrate is reduced [15,16]. Thus, as the number of metabolically active cells decreases, the glo signal decreases proportionally. The latter assay (flor, in the following text) measures changes in membrane integrity that occur as a result of cell death (i.e., an increase in the number of dead cells is proportional to the increase in fluorescent signal, which results from the increased number of DNA-dye aggregates detected due to the loss of cell membrane integrity) . Using the two real-time assays in a multiplexed mode, we measured in a single run the changes in cell viability and cell death in two cell lines induced by the Tox21 chemicals at 15 concentrations with a single well per concentration in a qHTS format (1536 well) after 0 (right after the chemical administration), 8, 16, 24, 32, and 40 hours of exposure. These sample times were selected for the screening convenience. The two cell lines used in this study were HEK293, a human embryonic kidney line, and HepG2, a human hepatocellular carcinoma cell line. Selection of these two cell lines was based on their extensive use in many other Tox21 reporter assays. In total, >11K concentration-response curves were generated at each time point using a single assay and a single cell line, with >266K concentration-response curves were generated in this study. The plate/well level data can be downloaded from the UNC Dataverse (https://dataverse.unc.edu/dataset.xhtml?persistentId=doi:10.15139/S3/12321) and are available in PubChem (https://www.ncbi.nlm.nih.gov/pcassay?term=tox21+real+time). The cytotoxicity kinetics data of mitomycin C in the HEK293 cell line using the glo assay technology (HEK293[glo], in the following text) is provided in Fig 1 as an example of data handling. Full paper
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