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BioAssay: AID 652044

PglD: Counter-screen assay Cytotoxicity Measured in Cell-Based System Using Plate Reader - 2164-02_Inhibitor_Dose_DryPowder_Activity

Assay Overview: Cell cultures of NIH 3T3 are prepared and compounds to be tested for toxicity are added to the culture medium. After a period of 48 hours exposure, cellular viability is measured using the Cell Titre Glo Assay (Promega). The assay measures ATP levels from the lysed cells using luciferase as the readout. In the case of toxicity, a lower luminescent signal will be observed. ..more
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 Tested Compounds
 Tested Compounds
All(16)
 
 
Active(2)
 
 
Inactive(14)
 
 
 Tested Substances
 Tested Substances
All(57)
 
 
Active(4)
 
 
Inactive(53)
 
 
AID: 652044
Data Source: Broad Institute (2164-02_Inhibitor_Dose_DryPowder_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2013-02-15

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 2
Related Experiments
AIDNameTypeComment
602402Broad Institute Inhibition of Glycoprotein Biosynthesis in Gram-Negative Pathogens Inhibitor Probe ProjectSummarydepositor-specified cross reference: Summary assay
602405PgID: DNTB colorimetric HTS to detect inhibitor of PgID Measured in Biochemical System Using Plate Reader - 2164-01_Inhibitor_SinglePoint_HTS_ActivityScreeningsame project related to Summary assay
624110PgID: DNTB colorimetric HTS to detect inhibitor of PgID Measured in Biochemical System Using Plate Reader - 2164-01_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
624257PgID: DNTB colorimetric HTS to detect inhibitor of PgID Measured in Biochemical System Using Plate Reader - 2164-01_Inhibitor_Dose_CherryPick_Activity_Set2Confirmatorysame project related to Summary assay
624403PgID: DNTB colorimetric HTS to detect inhibitor of PgID Measured in Biochemical System Using Plate Reader - 2164-01_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
652127PgID: DNTB colorimetric HTS to detect inhibitor of PgID Measured in Biochemical System Using Plate Reader - 2164-01_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
686993PgID: DNTB colorimetric HTS to detect inhibitor of PgID Measured in Biochemical System Using Plate Reader - 2164-01_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatorysame project related to Summary assay
743156PgID: DNTB colorimetric HTS to detect inhibitor of PgID Measured in Biochemical System Using Plate Reader - 2164-01_Inhibitor_SinglePoint_CherryPick_ActivityOthersame project related to Summary assay
Description:
Keywords: NIH3T3 cytotoxicity, Cell Titre Glo, viability assay

Assay Overview: Cell cultures of NIH 3T3 are prepared and compounds to be tested for toxicity are added to the culture medium. After a period of 48 hours exposure, cellular viability is measured using the Cell Titre Glo Assay (Promega). The assay measures ATP levels from the lysed cells using luciferase as the readout. In the case of toxicity, a lower luminescent signal will be observed.

Expected Outcome: Cytotoxic compounds will cause lower luminescence relative to controls. Non-cytotoxic compounds will not alter the luminescent signal relative to controls.
Protocol
NIH3T3 Cytotoxicity Assay
Day 0, cell grown in Triple flask (NUNC) to 95% confluence (TrypLE phenol free) and resuspended for dispensing at 125,000 cells / mL of DMEM, 10% FBS/Pen/Strep/L-Glutamine (Compact SelecT).
Day 1, plate cells 5000 per well in 40 uL media (DMEM/10% FBS/Pen/Strep/L-Glutamine); incubate in standard TC conditions (5% CO2; 95% humidity, 37C) for 24 hours (Compact SelecT).
Day 2, add 100 nL compound library retest request at dose into 40 uL assay volume in white, opaque Corning 8867 barcoded 384 well plates using a pin tool (HiRes Biosolutions). Pin 100 nL compound, 16 point dose curves starting at cytotoxic compounds was added to positive control wells to a final concentration of 16 uM (200 nL 4mM DMSO stock)
Incubate 48 hours at 37 degrees C in Liconic incubator, 95% humidity 5% CO2.
Day 3 vs. Day 4, remove plate from incubator to cool for 15 minutes to room temperature; add 20 uL 50% Promega CellTiterGlo (diluted 1:1 with PBS pH 7.4) with Thermo Combi.
Incubate at RT for 5 minutes.
Read on Perkin-Elmer Envision with US LUM settings for 0.1 sec per well.
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC; n=132) and positive control wells (PC; n=18) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (2)(Max_Concentration).
NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral controls (NC) is set to a normalized activity value of 0.
A normalized activity value of 100 is defined as (2)(NC).
A normalized activity value of -50 is defined as (0.5)(NC).
Experimental wells values were scaled to this range.
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.10.0.2) was applied.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)
PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.
PUBCHEM_ACTIVITY_SCORE:
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.
Note:
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Assay Type: Toxicity
Assay Cell Type: NIH 3T3
From ChEMBL:
Assay Type: Functional
Result Definitions
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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
3pAC50_MEqual to -1*log10(AC50)String
4Hill_SlopeThe slope at AC50Float
5S0_(%)The fitted activity value at zero concentrationFloat%
6Sinf_(%)The fitted activity value at infinite concentrationFloat%
7Num_PointsThe number of data points used to generate the plotInteger
8Max_Activity_(%)The maximum activity value observed, based on mean of replicates per concentrationFloat%
9Max_Activity_Conc_uMThe concentration at which the maximum activity is observedFloatμM
10Max_Concentration_uMMaximum valid test concentrationFloatμM
11Activity_at_0.0235uM_(%) (0.0235μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.05uM_(%) (0.05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.1uM_(%) (0.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_0.195uM_(%) (0.195μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_0.21uM_(%) (0.21μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_0.38uM_(%) (0.38μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_0.42uM_(%) (0.42μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_0.75uM_(%) (0.75μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_0.8uM_(%) (0.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20Activity_at_1.5uM_(%) (1.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
21Activity_at_1.6uM_(%) (1.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
22Activity_at_3uM_(%) (3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
23Activity_at_3.5uM_(%) (3.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
24Activity_at_6uM_(%) (6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
25Activity_at_6.8uM_(%) (6.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
26Activity_at_12uM_(%) (12μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
27Activity_at_13.5uM_(%) (13.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
28Activity_at_23.5uM_(%) (23.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
29Activity_at_26uM_(%) (26μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
30Activity_at_50uM_(%) (50μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH096549-01

Data Table (Concise)
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