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BioAssay: AID 655

Cellular Toxicity (caspase-3) Jurkat

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 caspase 3/7 of small molecules. This particular assay uses the Jurkat cell line which is derived from human T cell leukemia. ..more
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 Tested Compounds
 Tested Compounds
All(1335)
 
 
Active(48)
 
 
Inactive(1206)
 
 
Inconclusive(84)
 
 
 Tested Substances
 Tested Substances
All(1408)
 
 
Active(49)
 
 
Inactive(1271)
 
 
Inconclusive(88)
 
 
 Related BioAssays
 Related BioAssays
AID: 655
Data Source: NCGC (Caspase-3 Jurkat)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2007-04-12

Data Table ( Complete ):           Active    All
BioActive Compounds: 48
Description:
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Screening Centers Network [MLSCN]
National Institutes of Environmental Health Sciences [NIEHS]
National Toxicology Program [NTP]
Grant number: 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 caspase 3/7 of small molecules. This particular assay uses the Jurkat cell line which is derived from human T cell leukemia.
Protocol
NCGC Assay Protocol Summary:

The Caspase-Glo 3/7 assay (Promega) is a homogeneous method to measure caspase-3 and caspase-7 activities in cultured cells. The quantitation of caspase 3/7 activity is based on the cleavage of a peptide-aminoluciferin substrate. After caspase cleaves the substrate the free aminoluciferin is liberated and serves as the substrate of luciferase, which generates the light. The luminescent signal is proportional to amount of caspase 3/7 activity present.

Using the Caspase-Glo 3/7 assay, the amount of caspase 3/7 activity was measured in the Jurkat cell line with complete culture medium following compound treatment for 16 hours. The assay was performed in opaque white 1536-well plates. In the screen, tamoxifen and doxorubicin were used as positive controls. Library compounds were measured for their ability to cause the activation of caspase 3/7 in the cell line, as reflected by an increase in luminescence signal, in a concentration-dependent manner. Data were normalized to the controls for basal activity (DMSO only) and 100% stimulation (100 uM tamoxifen). AC50 values were determined from concentration-response data modeled with the standard Hill equation.

qHTS protocol for caspase 3/7 assay in Jurkat cells
[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 0.23 uM to 100 uM & Doxorubicin 7.0 pM to 100 uM
4. Time; 16 hr; 37#C incubation
5. Reagent; 5 uL; Caspase-Glo 3/7 reagent
6. Time; 20 min; Room Temperature
7. Detection; Luminescence; Viewlux plate reader

Keywords: caspase 3/7, apoptosis, Jurkat cell line, luminescence, NTPHTS, NTPHTS_NCGC, NIEHS, EPA, DSSTox, MLSMR, MLSCN, NIH Roadmap, qHTS, NCGC
Comment
Compound Ranking:

1. Compounds are first classified based on type of titration curve, and quality of curve fit and efficacy as being active (complete curve and good quality fit, or partial curve, good quality fit and high efficacy), inactive (flat curve), or inconclusive (other).

2. Within the active compounds, compounds were ranked by efficacy and potency. Compounds with the highest combined efficacy and potency are assigned a score of 100 and lowest a score of 50. Less conclusive activators are assigned a score of 40, likely activators are assigned a score of 30, inconclusive compounds are assigned a score of 15, and inactive compounds are assigned a score of 0.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: activator, inactive, or inconclusive.String
2PotencyConcentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.String
3EfficacyMaximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.Float%
4Analysis CommentAnnotation/notes on a particular compound's data or its analysis.String
5Curve DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically signficant, but below 80% of control.String
6Fit_LogAC50The logarithm of the AC50 from a fit of the ratio data to the Hill equation.Float
7Fit_HillSlopeThe Hill slope from a fit of the ratio data to the Hill equation.Float
8Fit_R2R^2 fit value of ratio curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the ratio data to the Hill equation.Float
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the ratio data to the Hill equation.Float
11Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12ExcludedPointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
13Activity at 0.6 nM% Activity at given concentration.Float%
14Activity at 3 nM% Activity at given concentration.Float%
15Activity at 14.8 nM% Activity at given concentration.Float%
16Activity at 33 nM% Activity at given concentration.Float%
17Activity at 73.8 nM% Activity at given concentration.Float%
18Activity at 164.9 nM% Activity at given concentration.Float%
19Activity at 368.7 nM% Activity at given concentration.Float%
20Activity at 824.5 nM% Activity at given concentration.Float%
21Activity at 1.8 uM% Activity at given concentration.Float%
22Activity at 4.1 uM% Activity at given concentration.Float%
23Activity at 9.2 uM% Activity at given concentration.Float%
24Activity at 20.6 uM% Activity at given concentration.Float%
25Activity at 46.1 uM% Activity at given concentration.Float%
26Activity at 92.2 uM% Activity at given concentration.Float%
27Compound TypeNCGC designation for compound stage: 'qHTS Exploratory', 'NIHSMR', 'Compound Followup', 'Compound Verification', 'Probe Optimization'String
28Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

Data Table (Concise)
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