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BioAssay: AID 1554

MLPCN Ras selective lethality-BJeLR viability

Brent Stockwell, Columbia University, 614 Fairchild Center, MC 2406, 1212 Amesterdam Ave, New York, NY 10027, bs2189@columbia.edu, 212.854.2948 ..more
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 Tested Compounds
 Tested Compounds
All(303280)
 
 
Active(516)
 
 
Inactive(302762)
 
 
Inconclusive(2)
 
 
 Tested Substances
 Tested Substances
All(303344)
 
 
Active(517)
 
 
Inactive(302825)
 
 
Inconclusive(2)
 
 
 Related BioAssays
 Related BioAssays
AID: 1554
Data Source: Broad Institute (2013-01)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2009-03-17
Modify Date: 2009-06-19

Data Table ( Complete ):           Active    All
BioActive Compounds: 516
Depositor Specified Assays
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AIDNameTypeProbeComment
1674Broad Institute MLPCN Ras Selective Lethality Projectsummary1
1933Luminescence Cell-Based Dose Response HTS to Identify Compounds Cytotoxic to BJ-TERT RAS-Independent Fibroblastconfirmatory
1934Luminescence Cell-Based Dose Response HTS to Identify Compounds Cytotoxic to DRD Non-Viral Oncogenic Fibroblastconfirmatory
1935Luminescence Cell-Based Dose Response HTS to Identify Compounds Cytotoxic to BJ-TERT-LT-ST RAS-Independent Fibroblastconfirmatory
1936Luminescence Cell-Based Dose Confirmation HTS to Identify Compounds Cytotoxic to BJeLR RAS-Dependent Fibroblastconfirmatory
2607Fluorescence Cell-Based Dose Response to Characterize Compounds Cytotoxic to Non-Viral Oncogenic DRD Fibroblasts.confirmatory
2608Fluorescence Cell-Based Dose Response to Characterize Compounds Cytotoxic to RAS-Dependent BJeLR Fibroblastsconfirmatory
2609Fluorescence Cell-Based Dose Response to Characterize Compounds Cytotoxic to RAS-Dependent BJ-TERT-LT-ST Fibroblasts.confirmatory
2610Fluorescence Cell-Based Dose Response to Characterize Compounds Cytotoxic to RAS-Dependent BJeLR Fibroblastconfirmatory
2611Fluorescence Cell-Based Dose Response to Characterize Compounds Cytotoxic to RAS-Dependent BJ-TERT Fibroblasts.confirmatory
2631Fluorescence Cell-Based Dose Response to Characterize Compounds Cytotoxic to RAS-Dependent BJ-TERT-LT-ST Fibroblastconfirmatory
2633Fluorescence Cell-Based Dose Response to Characterize Compounds Cytotoxic to Non-Viral Oncogenic DRD Fibroblastconfirmatory
2635Fluorescence Cell-Based Dose Response to Characterize Compounds Cytotoxic to RAS-Dependent BJ-TERT Fibroblastconfirmatory
Description:
Broad Institute MLPCN Ras Selective Lethality Project

Project ID: 2013

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

Primary Collaborators:

Brent Stockwell, Columbia University, 614 Fairchild Center, MC 2406, 1212 Amesterdam Ave, New York, NY 10027, bs2189@columbia.edu, 212.854.2948

Dan Zaharevitz, NCI Science Officer, zaharevd@mail.nih.gov

Project Overview:

The goal of the project is to identify small molecules in the MLSMR and related compound synthesized through follow-up chemistry that are selectively lethal to tumor cells expressing RAS oncogenes. These probes will not necessarily interact with the RAS pathway, but may induce cell death by affecting cellular processes that, in conjunction with the activation of the RAS pathway, selectively kill the tumor cells.

Assay Overview:

Cell viability of BJeLR (BJ fibroblasts transformed with hTERT, genomic SV40 LT and ST oncoproteins, and oncogenic HRASV12). Using CellTiterGlo, which measures the amound of intracellular ATP from living 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 luminescence signal due to fewer live cells. Specificity will be determined in future counterscreening.
Protocol
Taken from 2013-01-A01-07:
Positive control: RSL3, from Stockwell lab, 32 mg, assigned BRD-K56411643-001-01-8

Cell Passage
1. BJeLR cells (Stockwell lab) are normally maintained in 35mL of media in a T175 cell culture flask, which is kept in a TC incubator at 95% humidity, 5% CO2, 37 degrees C.
2. To passage, harvest the cells by first aspirating the media and rinsing the flask with 10 mL PBS. Aspirate the PBS and add 5 mL trypsin to the flask. Incubate with the trypsin for 5 minutes in the TC hood (22 degrees C).
3. Add 8 mL of media to the trypsin. Dislodge the cells from the bottom of the flask by pipeting. Remove an aliquot of cells for counting. Cell density should be between 1.5 and 2 x 106/mL.
4. Transfer 4.5 x 10^6 (~2 mL) of the 13 mL cells in trypsin/media to 33 mL fresh media in a T175 for harvest at ~90% confluence two days later, or 1.5 x 10^6 (~1 mL)34 mL fresh media for harvest three days later.
5. Lines are stopped at P20 and restarted from an earlier frozen aliquot.

Assay Protocol
Day -1
1. Harvest the cells by first aspirating the media and rinsing the flask with 10 mL PBS. Aspirate the PBS and add 5 mL trypsin to the flask. Incubate with the trypsin for 5 minutes in the TC hood (22 degrees C).
2. Add 8 mL of media to the trypsin. Dislodge the cells from the bottom of the flask by pipeting. Remove an aliquot of cells for counting.
3. Add media to adjust the concentration of the cells to 40,000/mL and add a sterile stir bar to the flask.
4. Plate the cells to 384-well plates using the Combi (walkup room), transferring 25 uL to each well, for a total of 1,000 cells/well. Keep the cell flask on a stir plate, gently spinning, to maintain a uniform cell suspension during the plating operation.
5. Incubate the plates overnight in a TC incubator at 95% humidity, 5% CO2, 37 degrees C.
Day 1
6. Transfer the plates to an online Liconic incubator at 95% humidity, 5% CO2, 37 degrees C.
7. Pin with 50 nL compound (BL2+ system, one dip) using 25nL head programmed to deliver 50 nL.
8. Return plates to the Liconic incubator at 95% humidity, 5% CO2, 37 degrees C for overnight incubation.
Day 2
9. Transfer the plates to offline TC incubators, 95% humidity, 5% CO2, 37 degrees C. (This step is done to free up working space in the Liconic incubator.)
Day 3
10. Move plates to racks in a Liconic incubator (on GS system), 95% humidity, 5% CO2, 37 degrees C. Load plate racks so that all plates will be pulled for the read run in the same order that they were pinned.
11. Initiate the automation protocol to start the read run. Plates are moved from the Liconic incubator to the GS carousel at normal room conditions (ambient humidity, 22 degrees C). Plates are allowed to cool on the carousel for 30 minutes, lids on.
12. Each plate is delided and 25 uL Cell Titer Glo (prepared as 1:3 dilution) is added to each well using the Combi.
13. Plates are incubated an additional 10 minutes in the carousel without lids.
14. Plates are read with the Envision, luminescence detection, read time = 0.1 second.
15. Plates are relided and discarded.
Comment
This assay belongs to specific project (2013, MLPCN Ras selective lethality) at the Broad Institute of MIT and Harvard

Activity_Score: plate effect corrected, background subtracted % activity average of two replicates

Activity_Outcome = 1
Activity_Score inhibition <50%.
Activity_Outcome = 2
Activity_Score inhibition >50%.
Activity_Outcome = 3
Wells where Activity_Score > 50% presumably due to uncorrected plate effects
Activity_Outcome = 4
NA
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1BROAD_SCREENING_RUN_ID_REPLICATE_ABThis is the unique internal ID given to each screening run at the Broad Institute, corresponding to replicates A & B.String
2BROAD_SCREENING_RUN_ID_REPLICATE_CDThis is the unique internal ID given to each screening run at the Broad Institute, corresponding to replicates C & D.String
3REPRODUCIBILITY_COSINE_TRANSFORMA measure of how well the activity reproduced across the two
samples. Computed as the absolute value of the cosine between the "replicate vector"
(ScoreA, ScoreB ---as well as ScoreC and/or ScoreD where applicable) and the vector (1, 1) representing perfect reproducibility.
NULL will appear in this column if a sample was not run in duplicate or if the data produced by one of the replicates was Invalid
Float
4REPLICATE_A_ACTIVITY_SCOREThe calculated activity score for the sample A, Null here denotes data was not used or was not producedFloat%
5REPLICATE_B_ACTIVITY_SCOREThe calculated activity score for the sample B, Null here denotes data was not used or was not producedFloat%
6REPLICATE_C_ACTIVITY_SCOREThe calculated activity score for the sample C, Null here denotes data was not used or was not producedFloat%
7REPLICATE_D_ACTIVITY_SCOREThe calculated activity score for the sample D, Null here denotes data was not used or was not producedFloat%
8DATE_SUBMITTEDDate data submitted to pubchemString
Additional Information
Grant Number: MH084117-01

Data Table (Concise)
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