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BioAssay: AID 720594

HepG2 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-02_Inhibitor_Dose_DryPowder_Activity_Set3

Assay Overview: The cytotoxcity assay uses wild-type HepG2 cells from ATCC test for cytotoxicity. The assay measures cellular ATP levels as a surrogate marker of cell viability with Pekin-Elmer CellTiter-Glo. Compound are tested at the same concentrations used in the primary assay. ..more
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 Tested Compounds
 Tested Compounds
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Active(1)
 
 
Inactive(41)
 
 
 Tested Substances
 Tested Substances
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Active(1)
 
 
Inactive(42)
 
 
AID: 720594
Data Source: Broad Institute (7071-02_Inhibitor_Dose_DryPowder_Activity_Set3)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2013-08-27
Hold-until Date: 2014-07-18
Modify Date: 2014-07-19

Data Table ( Complete ):           Active    All
BioActive Compound: 1
Depositor Specified Assays
AIDNameTypeComment
623900Broad Institute High Throughput Screen for Modulators of PAS/Co-activator Interactions Inhibitor Probe ProjectsummarySummary assay
Description:
Keywords: Cytotoxicity Assay, luminescence, CellTiter-Glo, HepG2


Assay Overview: The cytotoxcity assay uses wild-type HepG2 cells from ATCC test for cytotoxicity. The assay measures cellular ATP levels as a surrogate marker of cell viability with Pekin-Elmer CellTiter-Glo. Compound are tested at the same concentrations used in the primary assay.


Expected Outcome: Compounds identified as actives were toxic to cells at a compound concentration less than 10 uM. Activity in the assay leads to a reduction in cellular ATP levels which correlates with a decreased luminescence signal from the read reagent (CellTiter-Glo) and indicates cytotoxicity. Compounds that exhibit no cytotoxicity at IC50>30 uM will be prioritized for additional studies.
Protocol
Mammalian Cytotoxicity Assay Panel SOP


Assay Protocol:
Day 0, HepG2 cells were grown in Triple flasks (NUNC) to ~95% confluence (TrypLE Phenol Red free) and resuspended for dispensing at 125,000 cells/mL of DMEM, 10% FBS/Pen/Strep/L-Glutamine (using the TAP Compact SelecT automated cll culture system).

Day 1: Plate cells @5000 per well in 40 microL media (DMEM/10% FBS/Pen/Strep/L-Glutamine) using Corning 8867BC 384 well plates; incubate in standard TC conditions (5% CO2; 95% humidity, 37 degrees C) for 24 hours (Compact SelecT).

Day 2: Add 100 nL compound per well at dose into 40 uL assay volume using a pin tool (CyBi Well). Pin 100 nL cytotoxic compounds, mitoxantrone to positive control wells to a final concentration of 10 microM (100 nL 6.4 mM DMSO stock). Incubate for 72 hours at 37 degrees C in Liconic incubator, 95% humidity 5% CO2.

Day 4: Remove plate from incubator, cool for 15 minutes to room temperature; add 20 microL 50% Promega CellTiter-Glo (diluted 1:1 with PBS, pH 7.4) with Thermo Combi.
Incubate at RT for 5 minutes.

Read plates on a Perkin-Elmer EnVision plate reader with standard luminescence settings for 0.1 sec per well.
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC; n=104) and positive control wells (PC; n=20) 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.
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_0.000046uM_(%) (4.6e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.0001uM_(%) (0.0001μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.000195uM_(%) (0.000195μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_0.00038uM_(%) (0.00038μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_0.00075uM_(%) (0.00075μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_0.0015uM_(%) (0.0015μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_0.003uM_(%) (0.003μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_0.006uM_(%) (0.006μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_0.012uM_(%) (0.012μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20Activity_at_0.0235uM_(%) (0.0235μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
21Activity_at_0.05uM_(%) (0.05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
22Activity_at_0.1uM_(%) (0.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
23Activity_at_0.195uM_(%) (0.195μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
24Activity_at_0.38uM_(%) (0.38μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
25Activity_at_0.8uM_(%) (0.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
26Activity_at_1.6uM_(%) (1.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
27Activity_at_3uM_(%) (3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
28Activity_at_6uM_(%) (6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
29Activity_at_12uM_(%) (12μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
30Activity_at_26uM_(%) (26μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: R21 NS067624

Data Table (Concise)
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