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BioAssay: AID 540

Cell Viability - N2a

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 N2a cell line which is derived from mouse neuroblastoma. ..more
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 Tested Compounds
 Tested Compounds
All(1335)
 
 
Active(124)
 
 
Inactive(1114)
 
 
Inconclusive(105)
 
 
 Tested Substances
 Tested Substances
All(1408)
 
 
Active(131)
 
 
Inactive(1167)
 
 
Inconclusive(110)
 
 
 Related BioAssays
 Related BioAssays
AID: 540
Data Source: NCGC (cell-viability-n2a)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2006-11-30

Data Table ( Complete ):           Active    All
BioActive Compounds: 124
Description:
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 N2a cell line which is derived from mouse neuroblastoma.
Protocol
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 N2a 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 N2a cellular assay
[Step] [Parameter] [Value] [Description]
1. Reagent; 5 uL; 1500 N2a cells/well
2. Time; 5 hr; 37 oC 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; 37 oC incubation
6. Reagent; 5 uL; CellTiter Glo reagent
7. Time; 20 min; Room Temperature
8. Detection; Luminescence; Viewlux plate reader

Keywords: cell viability, cytotoxicity, N2a 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 (probable cytotoxic; 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 (cytotoxic) 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. Inactive compounds are assigned a score of 0, and inconclusive compounds are assigned a score of 25.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: cytotoxic, 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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