MITF: Counter assay: A375 proliferation Measured in Cell-Based System Using Plate Reader - 2084-03_Inhibitor_Dose_CherryPick_Activity_Set2
The confirmed modulators of MITF activity will be tested for MITF dependence in a melanoma cell line, A375, which proliferate at the normal rate when MITF levels are reduced with RNA interference methods and expresses MITF at very low levels compared to other melanoma cell lines (i.e. MITF independent melanoma). Cells will be treated with compounds at mulitple doses for 24 hours. Cytoxicity will be measured using cellular ATP levels as a readout. The Promega Cell Titer Glo reagent will be used to measure these ATP levels. ..more
BioActive Compounds: 266
The confirmed modulators of MITF activity will be tested for MITF dependence in a melanoma cell line, A375, which proliferate at the normal rate when MITF levels are reduced with RNA interference methods and expresses MITF at very low levels compared to other melanoma cell lines (i.e. MITF independent melanoma). Cells will be treated with compounds at mulitple doses for 24 hours. Cytoxicity will be measured using cellular ATP levels as a readout. The Promega Cell Titer Glo reagent will be used to measure these ATP levels.
Expected Outcome:A chemical modulator of MITF activity should decrease proliferation of MITF dependent cells but not a cell line that is not dependent upon MITF. Since A375 is not dependent upon MITF, active compounds should not alter A375 cell viability or proliferation. In addition, compounds that are general cytotoxins will impact A375 viability and be eliminated as a probe candidate.
- On day 1, plate cells 3000 per well in 30 uL phenol red free DMEM + 10% fetal bovine serum
- On day 2, pin 100 nL into 30 microL assay volume in white, opaque 384 plates. (addition of positive control requires sentinel pinning)
- Incubate 24 hours at 37 deg C
- On day 3, add 20 microL 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) and positive control wells (PC) were included on every plate.
EXPECTED OUTCOME: Active compounds were those which resulted in a dose-dependent activity change in either increasing or decreasing direction; only samples which were inactive were deemed interesting for further study.
ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was 35 uM and the active concentration limit (AC_limit) was set to equal (Max_Concentration) + (0.5Log).
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: The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata (v7.0.3) was applied to the normalized plate data.
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)