Primary human melanocyte cytotoxicity assay Measured in Cell-Based System Using Plate Reader - 2084-06_Inhibitor_Dose_DryPowder_Activity
Primary human melanocytes are distinct from melanoma cells and cell lines. They represent a population of cells that are dependent upon MITF for survival and a small minority of cells that are not dependent upon MITF. Therefore, compounds that inhibit MITF should have an effect on the viability of normal melanocytes. Cells will be treated with compound at dose for 24 hours. Cellular viability will be assessed using the Cell Titer Glo reagent from Promega that measures cellular ATP levels in a luminescence-based assay. ..more
BioActive Compounds: 10
Keywords: primary human melanocytes, MITF, cellular viability, melanoma
Primary human melanocytes are distinct from melanoma cells and cell lines. They represent a population of cells that are dependent upon MITF for survival and a small minority of cells that are not dependent upon MITF. Therefore, compounds that inhibit MITF should have an effect on the viability of normal melanocytes. Cells will be treated with compound at dose for 24 hours. Cellular viability will be assessed using the Cell Titer Glo reagent from Promega that measures cellular ATP levels in a luminescence-based assay.
Expected Outcome: Compounds that reduce viability or do not impact viability will be acceptable as a probe. Therefore, an increase, decrease or no alteration of signal is allowed.
Primary melanocytes are isolated from clinical skin samples. Skin tissue is lightly dissociated and cultured in media that favors melanocyte proliferation. Once homeogeneous, the primary human melanocytes are expanded in TIVA media (see below). Cells are plates at 4,000 cells per well in 30 ul media in a Corning 8867 barcoded plate (white, opaque). The following day, 100 nL of compound is added per well. Treatment with compound occurs for 24 hours and cell viability is measured with CellTiter Glo (Promega).
TIVA media: Ham's F-10 (Cellgro), 7% FBS, 1% penicillin/streptomycin, 2mM glutamine (Cellgro), 100 uM 3-isobutyl-1-methylxanthine (IBMX), 50 ng/ml 12-o-tetradecanoylphorbol 13-acetate (TPA), 1mM dibutyryl-cAMP (dbcAMP) (Sigma) and 1 microM sodium vanadate
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 Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
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
Assay Format: Cell-based
Assay Type: Toxicity
Assay Format: Cell-based
Assay Type: Functional
* Activity Concentration. ** Test Concentration.
Data Table (Concise)