MITF Orthogonal Assay: MALME3 CTG Assay Measured in Cell-Based System Using Plate Reader - 2084-04_Inhibitor_Dose_DryPowder_Activity_Set2
Some melanomas require MITF for survival and proliferation and other cell lines do not require MITF (i.e. MITF independent). The confirmed modulators of MITF activity will be tested for MITF dependence in another melanoma cell line, MALME3, which do not proliferate when MITF levels are reduced with RNA interference methods. Cells will be pinned with compounds at multiple doses and cell viability will be tested after 24 hours of treatment with Promega's Cell Titer Glo reagent. Cellular ATP levels will be measured as a surrogate marker of cell viability. ..more
BioActive Compounds: 83
Keywords: cytotoxicity, melanoma, MITF, MALME3 cells, luminescence
Some melanomas require MITF for survival and proliferation and other cell lines do not require MITF (i.e. MITF independent). The confirmed modulators of MITF activity will be tested for MITF dependence in another melanoma cell line, MALME3, which do not proliferate when MITF levels are reduced with RNA interference methods. Cells will be pinned with compounds at multiple doses and cell viability will be tested after 24 hours of treatment with Promega's Cell Titer Glo reagent. Cellular ATP levels will be measured as a surrogate marker of cell viability.
Expected Outcome: A true chemical modulator of MITF activity should decrease proliferation of MITF-dependent cells, such as MALME3, but not a cell line that is not dependent upon MITF, like A375. Active compounds in the MALME3 assay will lead to a decrease in luminescence signal.
- On day 1, plate cells 3000 per well in 30 uL phenol-red free (IMDM/10% FBS)
- On day 2, pin 100 nL into 30 uL assay volume in white, opaque Corning 8867 384 plates. (will also require sentinel pinning of parthenolide control)
- Incubate 24-36 hours at 37 degrees Celsius.
- On day 3, add 20 uL 100% Promega Cell Titer glo with a Thermo Combi fluid transfer apparatus.
- Shake 15 seconds on "big bear" plate shaker.
- Incubate at room temperature for 5 minutes.
- Read on Envision with standard luminescence settings
PRESENCE OF CONTROLS: Neutral control wells (NC; n=104) and positive control wells (PC; n=20) 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 (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: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) 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)