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BioAssay: AID 720585

A549 Cytoxocity Assay Measured in Cell-Based System Using Plate Reader - 7071-06_Inhibitor_Dose_DryPowder_Activity

Assay Overview: The assay uses A549 cells obtained from ATCC to test for cytotoxicity at the same concentrations used to confirm compound activity in the primary screen. Cellular ATP levels are measured as a surrogate of cell viability with the luminescence reagent, CellTiter-Glo (Promega). ..more
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Active(1)
 
 
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AID: 720585
Data Source: Broad Institute (7071-06_Inhibitor_Dose_DryPowder_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2013-08-26
Hold-until Date: 2014-08-21
Modify Date: 2014-08-21

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compound: 1
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743349BioA: Antitubercular activities in wild type Mtb (plus biotin) Measured in Cell-Based System Using Plate Reader - 2163-17_Inhibitor_SinglePoint_DryPowder_ActivityOthersame project related to Summary assay
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743354HEK293 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-01_Inhibitor_Dose_DryPowder_Activity_Set12Confirmatorysame project related to Summary assay
743356A549 Cytotoxicity Assay Measured in Cell-Based System Using Plate Reader - 7071-06_Inhibitor_Dose_DryPowder_Activity_Set10Confirmatorysame project related to Summary assay
743357Coupled Fluorescence Displacement Assay 20-point dose Measured in Biochemical System Using Plate Reader - 2163-12_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
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Description:
Keywords: Cytotoxicity Assay, A549, CellTiter-Glo, luminescence

Assay Overview: The assay uses A549 cells obtained from ATCC to test for cytotoxicity at the same concentrations used to confirm compound activity in the primary screen. Cellular ATP levels are measured as a surrogate of cell viability with the luminescence reagent, CellTiter-Glo (Promega).


Expected Outcome: Compounds identified as hits will be 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 (Promega's CellTiter-Glo) and indicates cytotoxicity. Compounds that exhibit no cytotoxicity at an IC50 >30 uM will be prioritized for additional studies.
Protocol
Assay Protocol:
Day 0, A549 cells grown in Triple flask (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 cell culture system).
Day 1: Plate cells at 5000 per well in 40 uL media (DMEM/10% FBS/Pen/Strep/L-Glutamine) using Corning 8867BC 384 well plates; incubate in standard TC conditions (5% CO2; 95% humidity, 37 deg 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 uM (100 nL 4 mM DMSO stock). Incubate for 72 hours at 37 deg 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 CellTiterGlo (diluted 1:1 with PBS, pH 7.4) with Thermo Combi.
Incubate at room temperature for 5 minutes.
Read plates on Perkin-Elmer EnVision with standard luminescence settings for 0.1 sec per well.
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC; n=32) and positive control wells (PC; n=32) 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 Minus Inhibitors' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
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): absACnn, the concentration at which the curve crosses threshold -50.0
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
1AbsAC50_Qualifier<, =, or >String
2AbsAC50_uM*The concentration at which the fitted curve passes activity threshold 50.FloatμM
3pAbsAC50_MEqual to -1*log10(AbsAC50).Float
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_0.195uM_(%) (0.195μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.38uM_(%) (0.38μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.8uM_(%) (0.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_1.6uM_(%) (1.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_3uM_(%) (3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_6uM_(%) (6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_12uM_(%) (12μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_26uM_(%) (26μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1R03MH096537-01

Data Table (Concise)
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