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BioAssay: AID 463211

Luminescent Cell-Based Counter Screen to Identify Non-Cytotoxic Compounds

At this stage in the project, the compounds have only been evaluated in biochemical (cell-free) assays. Since the compounds need to work in living cells, we need to evaluate the effect of compounds on cellular viability. HeLa cells are a human cell line and are used as a general indicator of cellular cytotoxicity. Cells are exposed to compounds for 48 hours with various concentrations of the compounds. After 48 hours, Promega Cell Titer Glo, which measures cellular ATP levels, is used to examine cellular viability in a luminescence-based assay. ..more
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 Tested Compounds
 Tested Compounds
All(35)
 
 
Active(11)
 
 
Inactive(24)
 
 
 Tested Substances
 Tested Substances
All(35)
 
 
Active(11)
 
 
Inactive(24)
 
 
AID: 463211
Data Source: Broad Institute (2053-02_INHIBITORS_DOSE-TITRATION_MLPCN-CHERRYPICK_TOXICITY)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-09-07
Modify Date: 2010-10-15

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 11
Related Experiments
AIDNameTypeComment
2629Fluorescence Polarization Cell-Free Homogeneous Primary HTS to Identify Inhibitors of the LANA Histone H2A/H2B InteractionScreeningdepositor-specified cross reference: Primary HTS
2659Summary of Broad Institute MLPCN Kaposi's Sarcoma Herpes Virus Latent Infection ProjectSummarydepositor-specified cross reference: Project summary
435023Fluorescence Polarization Cell-Free Homogeneous Dose Retest to Confirm Inhibitors of the LANA Histone H2A/H2B InteractionConfirmatorysame project related to Summary assay
463198Fluorescence Polarization Cell-Free Homogeneous Counter Screen to Identify Inhibitors of DNA IntercalationOthersame project related to Summary assay
588510Fluorescence Polarization Biochemical Secondary LANA-Nucleosome Assay_2053_04_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
Description:
Primary Collaborators:
Kenneth Kaye,Brigham and Women's,Boston MA,kkaye@rics.bwh.harvard.edu,617-525-4256
Chantal Beauchemin,Brigham and Women's,Boston MA,cbeauchemin@rics.bwh.harvard.edu,617-525-4256

Keywords: CellTiterGlo, HeLa, cytotoxicity, ATP

Assay Overview:
At this stage in the project, the compounds have only been evaluated in biochemical (cell-free) assays. Since the compounds need to work in living cells, we need to evaluate the effect of compounds on cellular viability. HeLa cells are a human cell line and are used as a general indicator of cellular cytotoxicity. Cells are exposed to compounds for 48 hours with various concentrations of the compounds. After 48 hours, Promega Cell Titer Glo, which measures cellular ATP levels, is used to examine cellular viability in a luminescence-based assay.
Protocol
Protocol:
HeLa cells are seeded at 3000 cells per well in 30 uL to 384-well plates. The following day, 100 nL of compound is added per well with the CyBio Vario pinning apparatus. Cells are incubated in the Liconic incubators for 48 hours at 37 degrees C. 30 uL of Cell Titer Glo (Promega) is added per well, shaken for 1 minute, incubated at room temperature for 10 minutes and then read on the Perkin Elmer Envision plate reader with standard luminescence parameters. Compounds which exhibit no cytotoxicity at 10microM and/or below will be prioritized for follow-up. Compounds will only be considered valid for subsequent studies if they do not kill mammalian cells.
Comment
Counterscreen Data Analysis

Negative control wells (NC) and positive control wells (PC) were included on every plate.
Active compounds result in decreased readout signal.

The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate NC wells was set to a normalized activity value of 0.
The median raw signal of the intraplate PC wells was set to a normalized activity value of -100.
Experimental wells were scaled to this range, giving an activity score as percent change in signal relative to the intraplate controls.

No plate pattern correction algorithm in Genedata was applied to the normalized plate data.

AC50 values were calculated using the curve fitting strategies in Genedata
Screener Condoseo (7.0.3). AC50 values were extrapolated up to 1 log
over the highest tested concentration.


PubChem Activity Score and Outcome

PUBCHEM_ACTIVITY_SCORE

Inactive compounds = 0
Active compounds = -10*Log(AC50)

PUBCHEM_ACTIVITY_OUTCOME

Activity_Outcome = 1 (inactive) when:
IC50 > 1 log over the highest tested concentration

Activity_Outcome = 2 (active) when:
IC50 <= 1 log over the highest tested concentration

Activity_Outcome = 3 (inconclusive) when:
Curve fitting strategy resulted in a constant fit with an activity score >30% but <70%
or
The fit was not valid due to poor fit quality.

Activity_Outcome = 4 (unspecified) when:
No usable data was obtained from the tested compound
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AC50_Qualifier'>', '=', or '<'String
2AC50_uM*the concentration at which activity reaches 50% of the maximumFloatμM
3Log_AC50_Mthe log of the EC50 (molar)Float
4Log_AC50_M_Standard_Errorthe standard error for the log of the EC50 valueFloat
5Hill_Slopethe slope at EC50Float
6S0the fitted activity level at zero concentrationFloat%
7SInfthe fitted activity level at infinite concentrationFloat%
8Num_Pointsthe number of data points included in the plotInteger
9Max_Activitythe maximum activity value observedFloat%
10Activity_at_15nM (0.015μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
11Activity_at_0.046uM (0.046μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.135uM (0.135μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.42uM (0.42μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_1.2uM (1.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_3.8uM (3.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_11uM (11μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_0.035mM (35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R21 NS061738-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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