Luminescence cell-based Retest at Dose assay to determine EWS/Fli1 dependent A673 mammalian cell cytotoxity Measured in Cell-Based System Using Plate Reader - 7014-03_Inhibitor_Dose_CherryPick_Activity
The assay is using an EWS/Fli dependent basal cell line called A673, the same cell line in the primary screen. Compounds inhibiting the activity of EWS/Fli should induce cell death in EWS/Fli-dependent manner. ..more
BioActive Compounds: 412
The assay is using an EWS/Fli dependent basal cell line called A673, the same cell line in the primary screen. Compounds inhibiting the activity of EWS/Fli should induce cell death in EWS/Fli-dependent manner.
Compounds identified as hits will be toxic to cells at a compound concentration less than 10 uM. Activity in the assay leads to a reduction in cellular ATP levels which correlates with a decreased luminescence signal from the read reagent (Promega's CellTiter-Glo) and indicates cytotoxicity. Compounds that exhibit cytotoxicity at =<30 uM will not be prioritized for additional studies.
Mammalian Cytotoxicity Assay Panel SOP
Goal: Standardize and consolidate work efforts of cytotoxicity testing for all probe projects at the Broad. All compounds will be tested in 2 different cell lines for 48 hours and must pass in all to be considered non-toxic. The panel of cell lines will be performed the first and third week of every month.
Day 0, A673 cells grown in Triple flask (NUNC) to ~95% confluence (TrypLE Phenol Red free) and resuspended for dispensing at 125,000 cells/mL of DMEM, 10% FBS/Pen/Strep/L-Glutamine (Compact SelecT).
Day 1: Plate cells @3000 per well in 40 microL media (DMEM/10% FBS/Pen/Strep/L-Glutamine) using Corning 8867BC 384 well plates; incubate in standard TC conditions (5% CO2; 95% humidity, 37 degrees C) for 24 hours (Compact SelecT).
Day 2: Add 100 nL compound per well at dose into 40 uL assay volume using a pin tool (CyBi Well). Pin 100 nL cytotoxic compounds, Mitoxantrone dihydrochloride(7mM) to positive control wells to a final concentration of 1microM (100 nL mM DMSO stock). Incubate for 72 hours at 37 degrees C in Liconic incubator, 95% humidity 5% CO2.
Day 5: Remove plate from incubator to cool for 15 minutes to room temperature; add 20 microL 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=36) and positive control wells (PC; n=36) 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 'Compounds' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate sample compound wells (SC) is set to a normalized activity value of 0.
A normalized activity value of 100 is defined as (2)(SC).
A normalized activity value of -50 is defined as (0.5)(SC).
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): absACnn, the concentration at which the curve crosses threshold -35.0
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
Assay Format: Cell-based
Assay Cell Type: A673
Assay Format: Cell-based
Assay Type: Functional
* Activity Concentration. ** Test Concentration.
Data Table (Concise)