MITF Orthogonal Assay: SK-MEL-5 CTG Assay Measured in Cell-Based System Using Plate Reader - 2084-02_Inhibitor_Dose_DryPowder_Activity_Set4
The confirmed modulators of MITF activity will be tested for MITF dependence in SK-MEL-5 melanoma cells which do not proliferate when MITF levels are reduced with RNA interference methods. This is the parental cell line for the reporter cell line used in the primary assay. Cells will be pinned with compounds and cell viability will be measured using cellular ATP levels as a read out. Cells will be treated with multiple doses of the compounds. Cell will be treated for 24 hours and ATP will be measured with Promega's Cell Titer Glo reagent. ..more
BioActive Compounds: 42
Keywords: melanoma, MITF, luciferase, cell titer glo
The confirmed modulators of MITF activity will be tested for MITF dependence in SK-MEL-5 melanoma cells which do not proliferate when MITF levels are reduced with RNA interference methods. This is the parental cell line for the reporter cell line used in the primary assay. Cells will be pinned with compounds and cell viability will be measured using cellular ATP levels as a read out. Cells will be treated with multiple doses of the compounds. Cell will be treated for 24 hours and ATP will be measured with Promega's Cell Titer Glo reagent.
Expected Outcome: A true inhibitor of MITF activity should decrease proliferation of MITF-dependent cells, such as SK-MEL-5, but not a cell line that is not dependent upon MITF. A candidate probe will decrease cellular viability with an IC50 of less than 10 uM.
- On day 1, plate cells 3000 per well in 30 uL phenol red free media
- On day 2, pin 100 nL into 30 uL assay volume in white, opaque 384 plates. (will also require sentinel pinning)
- Incubate 24 hours at 37 degrees C
- On day 3, add 20 uL 100% Promega Cell Titer glo with Thermo Combi fluid transfer apparatus.
- Shake 15 seconds on "big bear" plate shaker.
- Incubate at RT for 5 minutes.
- Read on Envision with standard luminescence settings
PRESENCE OF CONTROLS: Neutral control wells (NC; n=104) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
The compounds were assayed in multiple independent instances using an identical protocol; each instance is called a 'test'. For each test, the following analysis was applied:
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.
Once the data for each test was processed, the test number was appended to all column headers in that test's data set. The individual test results were then aggregated as follows:
1. The final PUBCHEM_ACTIVITY_OUTCOME was set to:
a. '2' (active) when all test outcomes were '2' (active), or
b. '1' (inactive) when all test outcomes were '1' (inactive), or
c. '3' (inconclusive) when the test outcomes were mixed.
2. The final ACTIVE_CONCENTRATION (AC) was set as follows:
a. If the final PUBCHEM_ACTIVITY_OUTCOME = 2, AC was set as the mean of the constituent test active concentrations;
b. If the final PUBCHEM_ACTIVITY_OUTCOME = 1 or 3, AC was left empty.
3. The final PUBCHEM_ACTIVITY_SCORE was calculated based on the aggregated ACTIVE_CONCENTRATION, using the same logic described above for individual test scores.
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)