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BioAssay: AID 433

Cell Viability - HepG2

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 HepG2 cell line which is derived from hepatocellular carcinoma. ..more
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AID: 433
Data Source: NCGC (cell-viability-hepg2)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2006-06-19
Modify Date: 2007-11-19

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 50
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 HepG2 cell line which is derived from hepatocellular carcinoma.
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 HepG2 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 fo CellTiter Glo HepG2 cellular assay
[Step] [Parameter] [Value] [Description]
1. Reagent; 5 uL; 1500 HepG2 cells/well
2. Time; 5 hr; 37oC incubation
3. Compounds; 23 nL; 0.59 nM to 92 uM
4. Controls; 23 nL; Tamoxifen 0.34 uM to 100 uM & Doxorubicin 0.02 nM to 100 uM
5. Time; 40 hr; 37oC incubation
6. Reagent; 5 uL; CellTiter Glo reagent
7. Time; 20 min; Room Temperature
8. Detection; Luminescence; Viewlux plate reader

Keywords: cell viability, cytotoxicity, HepG2 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
Categorized Comment - additional comments and annotations
From PubChem:
Assay Cell Type: HEPG2
Result Definitions
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OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Activity DirectionIndicates direction of activity: inactive, decreasing, increasing.String
2Activity QualifierIndicates if AC50 is "=" to value or if it is infinite activity (i.e, inactive), ">".String
3Qualified AC50Qualified AC50; can be an IC50 or EC50.Float
4Log of AC50Log of AC50.Float
5Hill CoefficientHill slope of fitted curve.Float
6Curve R2R^2 fit value of curve. Closer to 1.0 equates to better Hill equation fit.Float
7Data TypeNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String
8Compound QCNCGC comment on compound QC: 'DPI QC', 'NCGC Purified'String
9Data Analysis QCNCGC comment on confidence of data: 'Curve Verfied', etcString
10NCGC CommentAnnotation/notes on a particular compound's data: e.g, 'cytotoxic', 'fluorescent artifact'String
11Curve Fit ModelCurve fitting model used.String
12Hill S0S0 of Hill EquationFloat
13Hill SinfSinf of Hill EquationFloat
14Hill dSdS of Hill EquationFloat
15Log AC50 Std ErrorStandard error for Log AC50.Float
16Excluded PointsIndividual points excluded from curve fitting.String
17Number of PointsNumber of valid points used for fitting.Integer
18Activity at 0.59nM% Activity at given concentration.Float%
19Activity at 2.95nM% Activity at given concentration.Float%
20Activity at 14.751nM% Activity at given concentration.Float%
21Activity at 0.033uM% Activity at given concentration.Float%
22Activity at 0.074uM% Activity at given concentration.Float%
23Activity at 0.165uM% Activity at given concentration.Float%
24Activity at 0.369uM% Activity at given concentration.Float%
25Activity at 1.844uM% Activity at given concentration.Float%
26Activity at 9.217uM% Activity at given concentration.Float%
27Activity at 20.61uM% Activity at given concentration.Float%
28Activity at 0.046mM% Activity at given concentration.Float%
29Activity at 0.092mM% Activity at given concentration.Float%
30Compound TypeNCGC designation for compound stage: 'qHTS ECL (Exploratory)', 'NIHSMR (MLSCN)', 'Compound Followup', 'Compound Verification', 'Probe Optimization'String

Data Table (Concise)
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