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BioAssay: AID 1935

Luminescence Cell-Based Dose Response HTS to Identify Compounds Cytotoxic to BJ-TERT-LT-ST RAS-Independent Fibroblast

Assay: Cell viability of BJ-TERT (BJ fibroblasts transformed with hTERT and genomic SV40 LT and ST oncoproteins.) Using CellTiterGlo, which measures the amount 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. ..more
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 Tested Compounds
 Tested Compounds
All(1155)
 
 
Active(307)
 
 
Inactive(182)
 
 
Inconclusive(666)
 
 
 Tested Substances
 Tested Substances
All(1155)
 
 
Active(307)
 
 
Inactive(182)
 
 
Inconclusive(666)
 
 
 Related BioAssays
 Related BioAssays
AID: 1935
Data Source: Broad Institute (2013-03_SELECTIVELYCYTOTOXIC_DOSE-TITRATION_MLPCN-CHERRYPICK)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2009-09-08

Data Table ( Complete ):           Active    All
BioActive Compounds: 307
Depositor Specified Assays
AIDNameTypeComment
1554MLPCN Ras selective lethality-BJeLR viabilityscreeningPrimary HTS
1674Broad Institute MLPCN Ras Selective Lethality ProjectsummaryProject Summary
Description:
Keywords: Ras, apoptosis, cancer, VDAC, oxidative cell death

Assay: Cell viability of BJ-TERT (BJ fibroblasts transformed with hTERT and genomic SV40 LT and ST oncoproteins.) Using CellTiterGlo, which measures the amount 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, which do not contain oncogenic HRAS, are working through a mechanism that is not of interest for the goals this project.
Protocol
Positive control: RSL3, from Stockwell lab, 32 mg, assigned BRD-K56411643-001-01-8; Erastin, Batch1: Sigma E7718 assigned BRD-A25004090-001-01-9 or Batch2: Calbiochem 329600, assigned BRD-A25004090-001-02-7

Cell Passage
1. BJ-TERT-LT/ST 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 and 1.5 x 10^6/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 33,000/mL and add a sterile stir bar to the flask.
4. Plate the cells to 384-well plates using the Combi (walkup room), transferring 30 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, or 100 nL using 100nL head as appropriate.
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 delidded and 30 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 relidded and discarded.
Comment
Dose Response Data Analysis:
32 Negative control (DMSO) 16 positive control 1 (Erastin), 16 positive control 2 (RSL3) were included on each plate.

All wells tested on a plate were background subtracted using the median of the negative controls well values on the same plate. All wells tested on a plate were scaled relative to the median value of the same-plate positive control wells, using only the control more active for the tested cell line. Plate pattern correction was not used.

IC50 values were calculated using the Smart Fit strategy of Genedata
Screener Condoseo (v6.0.1). EC50 values were extrapolated up to 1 log
over the highest tested concentration.

PUBCHEM_ACTIVITY_SCORE
Inactive compounds = 0
Active compounds = -10*Log(EC50)

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

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

Activity_Outcome = 3 (inconclusive)
Constant fit curve actvitity is >30% but <70%
or
Constant fit curve is labelled as invalid due to large fit error
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1EC50 Qualifier'>', '=', or '<'String
2EC50*the concentration whereupon perceived activity reaches 50% of the maximumFloatμM
3EC50 Standard Errorthe standard error for the calculated EC50 valueFloatμM
4S0the fitted activity level at zero concentrationFloat%
5SInfthe fitted activity level at infinite concentrationFloat%
6Hill Slopethe slope at EC50Float
7# of Points Fitthe number of data points included in the plotInteger
8Max. Activitythe maximum activity value observedFloat%
9Activity at 0.12uM (0.12μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
10Activity at 0.23uM (0.23μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
11Activity at 0.46uM (0.46μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity at 0.90uM (0.9μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity at 1.80uM (1.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity at 3.80uM (3.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity at 7.50uM (7.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity at 15.00uM (15μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

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

Data Table (Concise)
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