NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Screening Centers Network [MLSCN]
National Institutes of Environmental Health Sciences [NIEHS]
National Toxicology Program [NTP]

NCGC Assay Overview:
We have developed a 1536-well cell-based assay for quantitative high throughput screening (qHTS) against a number of cell lines to determine in vitro cytotoxicity of small molecules. This particular assay uses the Jurkat cell line (Clone E6-1) which is derived from the human T cell leukemia.

NCGC Assay Protocol Summary:

The CellTiter-Glo luminescent cell viability assay (Promega) is a homogeneous method to measure the number of viable cells in culture. The end point readout of this assay is based on quantitation of intracellular ATP, an indicator of metabolic activity, using the luciferase reaction. Luciferase catalyzes the oxidation of beetle Luciferin to oxyluciferin and light in the presence of ATP. The luminescent signal is proportional to amount of ATP present.

Using the CellTiter-Glo luminescent cell viability assay, the amount of cellular ATP was measured in the Jurkat cell line with complete culture medium following compound treatment for 40 hours. The assay was performed in opaque white Kalypsys 1536-well plates. In the screen, tamoxifen and doxorubicin were used as positive controls. Library compounds were measured for their ability to cause acute toxicity in the cell line, as reflected by a decrease in intracellular ATP levels, in a concentration-dependent manner. Data were normalized to the controls for basal activity (DMSO only) and 100% inhibition (100 uM tamoxifen). AC50 values were determined from concentration-response data modeled with the standard Hill equation.

qHTS protocol for CellTiter Glo Jurkat cellular assay
[Step] [Parameter] [Value] [Description]
1. Reagent; 5 uL; 5000 Jurkat cells/well
2. Compounds; 23 nL; 0.59 nM to 92 uM
3. Controls; 23 nL; Tamoxifen 0.34 uM to 100 uM & Doxorubicin 0.02 nM to 100 uM
4. Time; 40 hr; 37#C incubation
5. Reagent; 5 uL; CellTiter Glo reagent
6. Time; 20 min; Room Temperature
7. Detection; Luminescence; Viewlux plate reader

Keywords: cell viability, cytotoxicity, Jurkat cell line, luminescence, NTPHTS, NTPHTS_NCGC, NIEHS, EPA, DSSTox, MLSMR, MLSCN, NIH Roadmap, qHTS, NCGC

Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive

2. Within each curve class, compounds are ranked by potency