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BioAssay: AID 2610

Fluorescence Cell-Based Dose Response to Characterize Compounds Cytotoxic to RAS-Dependent BJeLR Fibroblast

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(434)
 
 
Active(260)
 
 
Inactive(77)
 
 
Inconclusive(97)
 
 
 Tested Substances
 Tested Substances
All(435)
 
 
Active(261)
 
 
Inactive(77)
 
 
Inconclusive(97)
 
 
 Related BioAssays
 Related BioAssays
AID: 2610
Data Source: Broad Institute (2013-05_SELECTIVELYCYTOTOXIC_DOSE-TITRATION_MLPCN-CHERRYPICK)
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 ):           Active    All
BioActive Compounds: 260
Depositor Specified Assays
AIDNameTypeComment
1554MLPCN Ras selective lethality-BJeLR viabilityscreeningPrimary HTS
1674Broad Institute MLPCN Ras Selective Lethality ProjectsummaryProject Summary
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 BJeLR 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 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 37C/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 at 544nM excitation, 590nM emission
Comment
Cherrypick Dose Data Analysis
Using the fluorescence value of each well, % growth inhibition (%GI) was calculated, using a positive control of media only = 100% growth inhibition and a negative control of DMSO treated cells.
% GI = 100 * [1- {(cmpd treated cells) - (media only)/(cells only) - (media only)}]

Normalized data for each compound was manually imported into Genedata Condoseo (v 7.0.3) from external sources and multiplied by -1 to fit curves between 0% (no inhibition) and -100% (complete inhibition). 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)
In cases where more than one sample was reported, the PUBCHEM_ACTIVITY_SCORE was the mean of the individual scores for each sample.



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.

In cases where more than one sample is reported, the PUBCHEM_ACTIVITY_OUTCOME was set to 3 (inconclusive) if the outcomes of the individual samples did not agree.
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1SAMPLE1_ACTIVITY_OUTCOME1=Inactive/2=Active/3=InconclusiveInteger
2SAMPLE1_ACTIVITY_SCORERelative activity score for PubchemInteger
3SAMPLE1_EC50_Qualifier'>', '=', or '<'String
4SAMPLE1_EC50_uMthe concentration at which activity reaches 50% of the maximumFloatμM
5SAMPLE1_Log_EC50_Mthe concentration whereupon perceived activity reaches 50% of the maximumString
6SAMPLE1_Log_EC50_M_Standard_Errorthe standard error for the calculated EC50 valueFloatM
7SAMPLE1_Hill_Slopethe slope at EC50Float%
8SAMPLE1_S0the fitted activity level at zero concentrationFloat%
9SAMPLE1_SInfthe fitted activity level at infinite concentrationFloat
10SAMPLE1_Num_Pointsthe number of data points included in the plotInteger
11SAMPLE1_Max_Activitythe maximum activity value observedFloat%
12SAMPLE1_Activity_at_0.104uM (0.104μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13SAMPLE1_Activity_at_0.208uM (0.208μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14SAMPLE1_Activity_at_0.417uM (0.417μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15SAMPLE1_Activity_at_0.833uM (0.833μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16SAMPLE1_Activity_at_1.667uM (1.667μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17SAMPLE1_Activity_at_3.333uM (3.333μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18SAMPLE1_Activity_at_6.667uM (6.667μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19SAMPLE1_Activity_at_13.333uM (13.333μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20SAMPLE2_ACTIVITY_OUTCOME1=Inactive/2=Active/3=InconclusiveInteger
21SAMPLE2_ACTIVITY_SCORERelative activity score for PubchemInteger
22SAMPLE2_EC50_Qualifier'>', '=', or '<'String
23SAMPLE2_EC50_uM*the concentration at which activity reaches 50% of the maximumFloatμM
24SAMPLE2_Log_EC50_Mthe log of the EC50 (molar)String
25SAMPLE2_Log_EC50_M_Standard_Errorthe standard error for the log of the EC50 valueFloatM
26SAMPLE2_Hill_Slopethe slope at EC50Float%
27SAMPLE2_S0the fitted activity level at zero concentrationFloat%
28SAMPLE2_Sinfthe fitted activity level at infinite concentrationFloat
29SAMPLE2_Num_Pointsthe number of data points included in the plotInteger
30SAMPLE2_Max_Activitythe maximum activity value observedFloat%
31SAMPLE2_Activity_at_0.407nM (0.000407μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
32SAMPLE2_Activity_at_0.814nM (0.000814μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
33SAMPLE2_Activity_at_1.628nM (0.001628μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
34SAMPLE2_Activity_at_3.255nM (0.003255μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
35SAMPLE2_Activity_at_6.510nM (0.00651μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
36SAMPLE2_Activity_at_13.021nM (0.013201μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
37SAMPLE2_Activity_at_26.042nM (0.026042μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
38SAMPLE2_Activity_at_0.052uM (0.052μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
39SAMPLE2_Activity_at_0.104uM (0.104μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
40SAMPLE2_Activity_at_0.208uM (0.208μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
41SAMPLE2_Activity_at_0.417uM (0.417μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
42SAMPLE2_Activity_at_0.833uM (0.833μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
43SAMPLE2_Activity_at_1.667uM (1.667μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
44SAMPLE2_Activity_at_3.333uM (3.333μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
45SAMPLE2_Activity_at_6.667uM (6.667μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
46SAMPLE2_Activity_at_13.333uM (13.333μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
47DATE_REPORTEDThe date the data was internally reportedString

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

Data Table (Concise)
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