Viability secondary screen, Ras selective lethality- DRD cells Measured in Cell-Based System Using Plate Reader - 2013-08_Inhibitor_Dose_DryPowder_Activity
Assay Overview: Cell viability of DRD (BJ fibroblasts transformed with SV40 small T oncoprotein, dominant negative p53, cyclin D1, a mutant form of CDK4, and HRASV12.). Using Alamar Blue, which is metabolized to a fluorescent product by live cells, the total amount of viable cells can be measured by luminescence readout. Cells are treated with compounds for 48 hours to allow detection of compounds that may be slower-acting. ..more
BioActive Compounds: 13
Keywords: Ras, apoptosis, cancer, VDAC, oxidative cell death
Assay Overview: Cell viability of DRD (BJ fibroblasts transformed with SV40 small T oncoprotein, dominant negative p53, cyclin D1, a mutant form of CDK4, and HRASV12.). Using Alamar Blue, which is metabolized to a fluorescent product by live cells, the total amount of viable cells can be measured by luminescence readout. Cells are treated with compounds for 48 hours to allow detection of compounds that may be slower-acting.
Expected Outcome: Compounds that are toxic to these transformed fibroblasts, either selectively due to the HRAS oncogene or due to nonspecific toxicity, will cause a loss of fluorescence signal due to fewer live cells.
730 ml DMEM with 4mM L-glutamine (Gibco # 11995)
210 ml M199 (Sigma #M7528)
150 ml heat inactivated Fetal Bovine Serum (Gibco # 26140-079)
10 ml Penicillin/Streptomycin (Gibco #15140-122)
1.Make Daughter plates
a.Transfer 148uL of BJ media to empty Daughter plates
b.Transfer 2ul of cmpd solution in Mother plates to media-filled Daughter plates (1:75 dilution) and mix thoroughly
2.Make Step-Daughter plates
*Both Daughter and Step-Daughter plates are the same Greiner #781270, 384 cone deep
a.Fill empty Step-Daughter plate with 75uL BJ media except col3 and col13
b.Transfer 150uL of hits from Daughter plate to col3 and col13
-We fill C3 through N3 and C13 through N13; the total number of primary hits in a single Step-Daughter plate is 24 (12 on col3 and 12 on col13)
c.Make 2-fold dilution series by transferring 75uL to the next column (from col3 to col12 and col13 to col22)
3.Seed cells & add compound to Assay plates
a.Prepare BJeH cell solution
b.Transfer 36uL of DRD cell solution to empty Assay plates; the concentration of BJeH cells in the Assay plate is 1000 cells/well
c.Transfer 4ul of cmpd solution from Step-Daughter plate to cell-seeded Assay plates (1:10 dilution a final concentration of cmpd is from 0.01ug/mL to 5.33 ug/ml in 2-fold dilution series)
4.Incubate cells for 48h at 37oC/ 5%CO2
5.Add alamar blue solution to the Assay plates
a.Transfer 10ul of 50% alamar blue in BJ growth media to the Assay plates
b.Incubate ~16 hours before reading plates
6. Read Fluorescence @ 544 nM excitation, 590 nM emission
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) 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 (HVC) was determined and the active concentration limit (AC_limit) was set to equal (10)(HVC).
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.
Activity_Outcome = 1 (inactive) when:
a) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
b) AC > AC_limit, or
c) 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
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 not valid due to poor fit quality.
PUBCHEM_ACTIVITY_SCORE = 0 when PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive)
PUBCHEM_ACTIVITY_SCORE = (-10)[Log(AC)], where AC is in molar, when PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
PUBCHEM_ACTIVITY_SCORE = 100 when the curve fit is constant and showing full inhibition at all tested concentrations.
Categorized Comment - additional comments and annotations
Assay Format: Cell-based
Assay Cell Type: BJ
Assay Type: Functional
* Activity Concentration. ** Test Concentration.
Data Table (Concise)