PglD: Counter-screen assay Cytotoxicity Measured in Cell-Based System Using Plate Reader - 2164-02_Inhibitor_Dose_DryPowder_Activity
Assay Overview: Cell cultures of NIH 3T3 are prepared and compounds to be tested for toxicity are added to the culture medium. After a period of 48 hours exposure, cellular viability is measured using the Cell Titre Glo Assay (Promega). The assay measures ATP levels from the lysed cells using luciferase as the readout. In the case of toxicity, a lower luminescent signal will be observed. ..more
BioActive Compounds: 2
Keywords: NIH3T3 cytotoxicity, Cell Titre Glo, viability assay
Assay Overview: Cell cultures of NIH 3T3 are prepared and compounds to be tested for toxicity are added to the culture medium. After a period of 48 hours exposure, cellular viability is measured using the Cell Titre Glo Assay (Promega). The assay measures ATP levels from the lysed cells using luciferase as the readout. In the case of toxicity, a lower luminescent signal will be observed.
Expected Outcome: Cytotoxic compounds will cause lower luminescence relative to controls. Non-cytotoxic compounds will not alter the luminescent signal relative to controls.
NIH3T3 Cytotoxicity Assay
Day 0, cell grown in Triple flask (NUNC) to 95% confluence (TrypLE phenol free) and resuspended for dispensing at 125,000 cells / mL of DMEM, 10% FBS/Pen/Strep/L-Glutamine (Compact SelecT).
Day 1, plate cells 5000 per well in 40 uL media (DMEM/10% FBS/Pen/Strep/L-Glutamine); incubate in standard TC conditions (5% CO2; 95% humidity, 37C) for 24 hours (Compact SelecT).
Day 2, add 100 nL compound library retest request at dose into 40 uL assay volume in white, opaque Corning 8867 barcoded 384 well plates using a pin tool (HiRes Biosolutions). Pin 100 nL compound, 16 point dose curves starting at cytotoxic compounds was added to positive control wells to a final concentration of 16 uM (200 nL 4mM DMSO stock)
Incubate 48 hours at 37 degrees C in Liconic incubator, 95% humidity 5% CO2.
Day 3 vs. Day 4, remove plate from incubator to cool for 15 minutes to room temperature; add 20 uL 50% Promega CellTiterGlo (diluted 1:1 with PBS pH 7.4) with Thermo Combi.
Incubate at RT for 5 minutes.
Read on Perkin-Elmer Envision with US LUM settings for 0.1 sec per well.
PRESENCE OF CONTROLS: Neutral control wells (NC; n=132) and positive control wells (PC; n=18) 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).
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)
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.
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.
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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)