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BioAssay: AID 2609

Fluorescence Cell-Based Dose Response to Characterize Compounds Cytotoxic to RAS-Dependent BJ-TERT-LT-ST Fibroblasts.

Brent Stockwell,Columbia University,bs2198@columbia.edu,212-854-2948,614 Fairchild Center, MC 2406, 1212 Amsterdam Ave, New York, NY 10027 ..more
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 Tested Compounds
 Tested Compounds
All(14)
 
 
Active(14)
 
 
 Tested Substances
 Tested Substances
All(14)
 
 
Active(14)
 
 
 Related BioAssays
 Related BioAssays
AID: 2609
Data Source: Broad Institute (2013-07_SELECTIVELYCYTOTOXIC_DOSE-TITRATION_MLPCN-DRYPOWDER)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2010-03-19
Hold-until Date: 2010-09-30
Modify Date: 2010-09-30

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 14
Related Experiments
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AIDNameTypeProbeComment
1554MLPCN Ras selective lethality-BJeLR viabilityScreening depositor-specified cross reference: Primary HTS
1674Broad Institute MLPCN Ras Selective Lethality ProjectSummary1 depositor-specified cross reference: Summary
1933Luminescence Cell-Based Dose Response HTS to Identify Compounds Cytotoxic to BJ-TERT RAS-Independent FibroblastConfirmatory same project related to Summary assay
1934Luminescence Cell-Based Dose Response HTS to Identify Compounds Cytotoxic to DRD Non-Viral Oncogenic FibroblastConfirmatory same project related to Summary assay
1935Luminescence Cell-Based Dose Response HTS to Identify Compounds Cytotoxic to BJ-TERT-LT-ST RAS-Independent FibroblastConfirmatory same project related to Summary assay
1936Luminescence Cell-Based Dose Confirmation HTS to Identify Compounds Cytotoxic to BJeLR RAS-Dependent FibroblastConfirmatory same project related to Summary assay
2275Luminescence Cell-Based Primary HTS to Measure Viability of BJeLR cellsScreening same project related to Summary assay
2607Fluorescence Cell-Based Dose Response to Characterize Compounds Cytotoxic to Non-Viral Oncogenic DRD Fibroblasts.Confirmatory same project related to Summary assay
2608Fluorescence Cell-Based Dose Response to Characterize Compounds Cytotoxic to RAS-Dependent BJeLR FibroblastsConfirmatory same project related to Summary assay
2610Fluorescence Cell-Based Dose Response to Characterize Compounds Cytotoxic to RAS-Dependent BJeLR FibroblastConfirmatory same project related to Summary assay
2611Fluorescence Cell-Based Dose Response to Characterize Compounds Cytotoxic to RAS-Dependent BJ-TERT Fibroblasts.Confirmatory same project related to Summary assay
2631Fluorescence Cell-Based Dose Response to Characterize Compounds Cytotoxic to RAS-Dependent BJ-TERT-LT-ST FibroblastConfirmatory same project related to Summary assay
2633Fluorescence Cell-Based Dose Response to Characterize Compounds Cytotoxic to Non-Viral Oncogenic DRD FibroblastConfirmatory same project related to Summary assay
2635Fluorescence Cell-Based Dose Response to Characterize Compounds Cytotoxic to RAS-Dependent BJ-TERT FibroblastConfirmatory same project related to Summary assay
493045Primary viability screen, Ras selective lethality- BJeLR cells Measured in Cell-Based System Using Plate Reader - 2013-05_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
493049Viability secondary screen, Ras selective lethality- DRD cells Measured in Cell-Based System Using Plate Reader - 2013-08_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
493050Viability secondary screen, Ras selective lethality- DRD cells Measured in Cell-Based System Using Plate Reader - 2013-08_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
493051Viability counterscreen, Ras selective lethality- BJeH cells Measured in Cell-Based System Using Plate Reader - 2013-06_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
493052Viability counterscreen, Ras selective lethality- BJeHLT cells Measured in Cell-Based System Using Plate Reader - 2013-07_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
493053Primary viability screen, Ras selective lethality- BJeLR cells Measured in Cell-Based System Using Plate Reader - 2013-05_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
493054Viability counterscreen, Ras selective lethality- BJeH cells Measured in Cell-Based System Using Plate Reader - 2013-06_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
493093Viability counterscreen, Ras selective lethality- BJeHLT cells Measured in Cell-Based System Using Plate Reader - 2013-07_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
540314Viability counterscreen, Ras selective lethality- BJeHLT cells Measured in Cell-Based System Using Plate Reader - 2013-07_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatory same project related to Summary assay
540315Viability secondary screen, Ras selective lethality- DRD cells Measured in Cell-Based System Using Plate Reader - 2013-08_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatory same project related to Summary assay
540316Viability counterscreen, Ras selective lethality- BJeH cells Measured in Cell-Based System Using Plate Reader - 2013-06_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatory same project related to Summary assay
540318Primary viability screen, Ras selective lethality- BJeLR cells Measured in Cell-Based System Using Plate Reader - 2013-05_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatory same project related to Summary assay
652070HT29 HRAS Target ID Apoptosis Mulitlpe Timepoints Caspase 3/7 Measured in Cell-Based System Using Plate Reader - 2156-21_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
652072LOX IMVI HRAS Target ID Apoptosis Mulitlpe Timepoints Caspase 3/7 Measured in Cell-Based System Using Plate Reader - 2156-22_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
Description:
Primary Collaborators(and laboratory where this assay was performed):
Brent Stockwell,Columbia University,bs2198@columbia.edu,212-854-2948,614 Fairchild Center, MC 2406, 1212 Amsterdam Ave, New York, NY 10027
Dan Zaharevitz,NCI Science Officer,zaharevd@mail.nih.gov

Keywords: Ras, apoptosis, cancer, VDAC, oxidative cell death

Assay: Cell viability of BJeLR (BJ fibroblasts transformed with hTERT, genomic SV40 LT and ST oncoproteins, and oncogenic HRASV12). Using Alamer 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.
Protocol
Growth medium:
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 BJeH-LT 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 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 approx. 16 hours before reading plates

6. Read Fluorescence at 544 nM excitation, 590 nM emission
Comment
Powder Dose Data Analysis

Normalized data for each compound was manually imported into Genedata Condoseo (v 7.0.3) from external sources, and dose response curves were fit using the available algorithms (Smart Fit, etc.).


The PUBCHEM_ACTIVITY_SCORE was set as follows:
inactive compounds -> 0
active compounds -> -10*log(EC50)


The PUBCHEM_ACTIVITY_OUTCOME was set as follows:

Activity_Outcome = 1 (inactive) when
EC50 > 1 log over the highest tested concentration

Activity_Outcome = 2 (active) when
EC50 <= 1 log over the highest tested concentration

Activity_Outcome = 3 (inconclusive) when
Curve fitting strategy resulted in a constant fit with activity >30% but <70% ('undefined' in Genedata)
or
The fit was not valid due to poor fit quality.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Assay Cell Type: BJ
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1EC50 Qualifier'>', '=', or '<'String
2EC50(uM)*the concentration whereupon perceived activity reaches 50% of the maximumFloatμM
3Log EC50(M)the log of the EC50 (molar)String
4Log EC50(M) Standard Errorthe standard error for the log of the EC50FloatM
5Hill Slopethe slope at EC50Float
6S0the fitted activity level at zero concentrationFloat%
7SInfthe fitted activity level at infinite concentrationFloat%
8Num. Pointsthe number of data points included in the plotInteger
9Max. Activitythe maximum activity value observedFloat%

* Activity Concentration.
Additional Information
Grant Number: MH084117-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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