Nitrite Measured in Cell-Based System Using Plate Reader - 2061-04_Inhibitor_Dose_DryPowder_Activity
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
BioActive Compounds: 7
Keywords: nitrite production, Griess reagent, INS-1E cells, cytokine-induced apoptosis
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.
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).
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.
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 = 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.
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
* Activity Concentration. ** Test Concentration.
Data Table (Concise)