A549 24 hour CellTiterGlo cytotoxicity Measured in Cell-Based System Using Plate Reader - 2142-02_Inhibitor_Dose_CherryPick_Activity
Assay Overview: The goal of this assay was to determine cytotoxicity in A549 cells after 24 hour treatment with compound. The assay uses a luminescent readout via the CellTiter Glo reagent (Promega, Madison, WI) and is based on measuring the ATP content within cells. The more viable cells there are in a well, the more ATP is available and subsequently leads to a higher fluorescent signal. ..more
BioActive Compounds: 25
Keywords: A549, cytotoxicity, cellTiterGlo, luminescence
Assay Overview: The goal of this assay was to determine cytotoxicity in A549 cells after 24 hour treatment with compound. The assay uses a luminescent readout via the CellTiter Glo reagent (Promega, Madison, WI) and is based on measuring the ATP content within cells. The more viable cells there are in a well, the more ATP is available and subsequently leads to a higher fluorescent signal.
Expected Outcome: Compounds which are cytotoxic will cause a drop in the luminescent signal and will be deprioritized from further consideration.
12.5 nL of compounds dissolved in DMSO at different concentrations, were transferred to a 1536 well clear bottom black assay plates (Greiner Bio # 782096-01-F) by ATS Acoustic Transfer System (EDC Biosystem), for a final top concentration of 20 microM. Four microL of A549 cells at 2.5 X 105 cells/mL in DMEM containing 10% FBS were dispensed by Multidrop Combi dispenser (Thermo Scientific) into to the plates containing the compounds and cultured at 37 degrees C, 95% humidity, and 5% CO2 for 24 hours, then 4 microL of CellTiter Glo cell viability assay reagent (Promega) was dispensed into the each well of the plates. The plates were then incubated for 30 minutes at 37 degrees C and 5% CO2 before they were read on ViewLux (Perkin Elmer).
PRESENCE OF CONTROLS: Neutral control wells (NC; n=192) 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).
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.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)