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BioAssay: AID 488870

Nitrite Measured in Cell-Based System Using Plate Reader - 2061-04_Inhibitor_Dose_DryPowder_Activity_Set3

Cytokine induced apoptosis leads to downstream activation of nitric oxide synthase (NOS). NOS generates nitrites which are then released into the culture media by cells. The Griess reagent is a colorimetric assay for nitrites, and nitrates that have been reduced to nitrites, with a low level of detection. INS-1E cells will be treated with cytokines for 48 hours in the presence of compounds. Nitrite levels will be measured by absorbance. ..more
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 Tested Compounds
 Tested Compounds
All(85)
 
 
Active(26)
 
 
Inactive(59)
 
 
 Tested Substances
 Tested Substances
All(86)
 
 
Active(27)
 
 
Inactive(59)
 
 
 Related BioAssays
 Related BioAssays
AID: 488870
Data Source: Broad Institute (2061-04_Inhibitor_Dose_DryPowder_Activity_Set3)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-10-22
Hold-until Date: 2011-06-13
Modify Date: 2011-06-13

Data Table ( Complete ):           Active    All
BioActive Compounds: 26
Depositor Specified Assays
AIDNameTypeComment
435007Summary of Broad Institute MLPCN Beta Cell Apoptosis Inhibition ProjectsummarySummary assay
Description:
Keywords: nitrite production, Griess reagent, INS-1E cells, cytokine-induced apoptosis

Assay Overview:
Cytokine induced apoptosis leads to downstream activation of nitric oxide synthase (NOS). NOS generates nitrites which are then released into the culture media by cells. The Griess reagent is a colorimetric assay for nitrites, and nitrates that have been reduced to nitrites, with a low level of detection. INS-1E cells will be treated with cytokines for 48 hours in the presence of compounds. Nitrite levels will be measured by absorbance.
Protocol
Protocol:
INS-1E cells were seeded at 10,000 cells/well using a Multidrop Combi (Thermo Labsystems) in clear 384-well tissue culture plates (Corning Life Sciences).

After overnight incubation, phenol-red free plating medium containing a combination of cytokines (10 ng/mL IL-1b, 50 ng/mL IFN-g, 25 ng/mL TNF-a) was added to every well. Using libraries of compounds dissolved in DMSO and a CyBi-Well pin-transfer robot (CyBio Corp.), 0.1 uL of each compound was added to the wells.

After treatment with cytokine and compounds for 48hr, 10 uL modified Griess reagent (1:1 mixture of 1% sulfanilamide in 30% acetic acid and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride in 60% acetic acid) was added to each well.

After 5-min incubation at room temperature, the absorbance at 540 nm was measured using an Envision plate reader (PerkinElmer).
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) 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 (HVC) was determined and the active concentration limit (AC_limit) was set to equal (10)(HVC).

NORMALIZATION:
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 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.7.0.3) 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.

PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when:
a) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
b) AC > AC_limit, or
c) 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

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 not valid due to poor fit quality.

PUBCHEM_ACTIVITY_SCORE:

PUBCHEM_ACTIVITY_SCORE = 0 when PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive)

PUBCHEM_ACTIVITY_SCORE = (-10)[Log(AC)], where AC is in molar, when PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
Scores relate to AC in this manner:

120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M

PUBCHEM_ACTIVITY_SCORE = 100 when the curve fit is constant and showing full inhibition at all tested concentrations.


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
3Log_AC50_MThe log of the molar AC50Float
4Log_AC50_M_Standard_ErrorThe standard error for the log of the molar AC50 valueFloat
5Hill_SlopeThe slope at AC50Float
6S0The fitted activity value at zero concentrationFloat%
7SinfThe fitted activity value at infinite concentrationFloat%
8Num_PointsThe number of data points used to generate the plotInteger
9Max_ActivityThe maximum activity value observed, based on mean of replicates per concentrationFloat%
10Max_Activity_Conc_uMThe concentration at which the maximum activity is observedFloatμM
11Activity_at_0.16uM (0.16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.3uM (0.3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.6uM (0.6μM**)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_2.6uM (2.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_5uM (5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_10uM (10μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_19.5uM (19.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: DP2 DK083048

Data Table (Concise)
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