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

MLSCN Grant: None

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 a human lymphoblastoid cell line (LYMP2-007) purchased from Coriell Institute for Medical Research.

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 lymphoblastoid cell line with complete culture medium following compound treatment for 24 hours. The assay was performed in opaque white Kalypsys 1536-well plates. In the screen, tetraoctylammonium bromide was used as positive control. 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 (92 uM tetraoctylammonium bromide). AC50 values were determined from concentration-response data modeled with the standard Hill equation.

qHTS protocol for CellTiter Glo lymphoblastoid cellular assay
[Step] [Parameter] [Value] [Description]
1. Reagent; 5 uL; 2500 lymphoblastoid cells/well
2. Time; 5 hr; 37 C incubation
3. Compounds; 23 nL; 0.59 nM to 92 uM
4. Controls; 23 nL; Tetraoctylammonium bromide 6.4pM to 92 uM
5. Time; 24 hr; 37 C incubation
6. Reagent; 5 uL; CellTiter Glo reagent
7. Time; 30 min; Room Temperature
8. Detection; Luminescence; Viewlux plate reader

Keywords: cell viability, cytotoxicity, lymphoblastoid cell line, luminescence, NTPHTS, NTPHTS_NCGC, NIEHS, 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. See data field "Curve Description".

2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.