AID	Name	Type	Comment
435007	Summary of Broad Institute MLPCN Beta Cell Apoptosis Inhibition Project	summary	Summary assay

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:
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).

PRESENCE OF CONTROLS: Neutral control wells (NC) 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' method in Genedata Assay Analyzer (v7.0.3):
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: The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata (v7.0.3) was applied to the normalized plate data.

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.

Grant Number: DP2 DK083048