Bookmark and Share
BioAssay: AID 504407

Counterscreen of A1 inhibitors measured by cytochrome c ELISA in CHL-1 wild type cells Measured in Biochemical System Using Plate Reader - 2045-18_Inhibitor_SinglePoint_DryPowder_Activity

The fate of cell survival versus apoptosis is determined by the balance of anti and pro-apoptotic proteins. Expression of activator BH3-only proteins, such as BIM or tBID, leads to downstream caspase activation and apoptosis. A1 can functionally bind to and sequester BIM or tBID. An A1 inhibitor causes the release of A1-bound BIM, which activates BAX/BAK, and leads to caspase activation and more ..
_
   
 Tested Compounds
 Tested Compounds
All(1)
 
 
Inactive(1)
 
 
 Tested Substances
 Tested Substances
All(1)
 
 
Inactive(1)
 
 
AID: 504407
Data Source: Broad Institute (2045-18_Inhibitor_SinglePoint_DryPowder_Activity)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2011-03-02
Hold-until Date: 2011-10-31
Modify Date: 2011-10-31

Data Table ( Complete ):           View All Data
Target
Tested Compound:
Related Experiments
Show more
AIDNameTypeProbeComment
2526Summary of Broad Institute MLPCN A1 Apoptosis ProjectSummary1 depositor-specified cross reference: Summary assay
2462Luminescence Cell-Based Primary HTS to Identify Inhibitors of A1 Apoptosis.Screening same project related to Summary assay
2465Luminescence Cell-Based Primary HTS to Identify Compounds which Inhibit A1 ApoptosisScreening same project related to Summary assay
2765Luminescence Cell-Based Dose Retest to Identify Inhibitors of A1 ApoptosisConfirmatory same project related to Summary assay
449754Luminescence Cell-Based Counterscreen to Identify Inhibitors of A1 Apoptosis (Bax-Bak knockout)Confirmatory same project related to Summary assay
449755Luminescence Cell-Based Counterscreen to Identify Inhibitors of A1 Apoptosis (HMC 1-8 low)Confirmatory same project related to Summary assay
449757Luminescence Cell-Based Secondary Screen to Identify Inhibitors of A1(alternate construct)Confirmatory same project related to Summary assay
449761Luminescence Cell-Based Counterscreen to Identify Inhibitors of A1 Apoptosis (non-primed)Confirmatory same project related to Summary assay
488858Inhibitors of A1-dependent Mouse Embryonic Fibroblasts Measured in Cell-Based System Using Plate Reader - 2045-01_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
488885Inhibitors of A1 in HMC1-8 Mcl1 Dependent Cells Measured in Cell-Based System Using Plate Reader - 2045-03_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
488891Secondary assay of A1 inhibitors in Mouse Embryonic Fibroblasts with alternate A1 construct Measured in Cell-Based System Using Plate Reader - 2045-05_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
488897Secondary assay of A1 inhibitors in Mouse Embryonic Fibroblasts with alternate pro-apoptotic inducer Measured in Cell-Based System Using Plate Reader - 2045-08_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
488898Inhibitors of Bcl2-A1 in Bax/Bak -/- Mouse Embryonic Fibroblasts Measured in Cell-Based System Using Plate Reader - 2045-02_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
488902Counterscreen of A1 inhibitors in wild-type Mouse Embryonic Fibroblasts Measured in Cell-Based System Using Plate Reader - 2045-04_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
488914Secondary assay of A1 inhibitors in Trichostatin A primed human MEWO cells Measured in Cell-Based System Using Plate Reader - 2045-07_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
488934Secondary assay of A1 inhibitors in A1 dependent Mel501 cells Measured in Cell-Based System Using Plate Reader - 2045-09_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
488948Counterscreen of A1 inhibitors in unprimed human MEWO cells Measured in Cell-Based System Using Plate Reader - 2045-06_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
504342Secondary assay of A1 inhibitors in Mouse Embryonic Fibroblasts with alternate A1 construct Measured in Cell-Based System Using Plate Reader - 2045-05_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
504343Counterscreen of A1 inhibitors in HMC1-8 Mcl1 dependent cells Measured in Cell-Based System Using Plate Reader - 2045-03_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
504344Counterscreen of A1 inhibitors in Bax/Bak -/- Mouse Embryonic Fibroblasts Measured in Cell-Based System Using Plate Reader - 2045-02_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
504345Inhibitors of A1-dependent Mouse Embryonic Fibroblasts Measured in Cell-Based System Using Plate Reader - 2045-01_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
504346Counterscreen of A1 inhibitors in unprimed human MEWO cells Measured in Cell-Based System Using Plate Reader - 2045-06_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
504347Inhibitors of A1-dependent Mouse Embryonic Fibroblasts Measured in Cell-Based System Using Plate Reader - 2045-01_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatory same project related to Summary assay
504348Counterscreen of A1 inhibitors in HMC1-8 Mcl1 dependent cells Measured in Cell-Based System Using Plate Reader - 2045-03_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
504353Counterscreen of A1 inhibitors in wild-type Mouse Embryonic Fibroblasts Measured in Cell-Based System Using Plate Reader - 2045-04_Inhibitor_Dose_DryPowder_Activity_Set4Confirmatory same project related to Summary assay
504354Counterscreen of A1 inhibitors in unprimed human MEWO cells Measured in Cell-Based System Using Plate Reader - 2045-06_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
504356Secondary assay of A1 inhibitors in Trichostatin A primed human MEWO cells Measured in Cell-Based System Using Plate Reader - 2045-07_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
504359Secondary assay of A1 inhibitors in Mouse Embryonic Fibroblasts with alternate pro-apoptotic inducer Measured in Cell-Based System Using Plate Reader - 2045-08_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
504360Secondary assay of A1 inhibitors in Mouse Embryonic Fibroblasts with alternate pro-apoptotic inducer Measured in Cell-Based System Using Plate Reader - 2045-08_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
504365Secondary assay of A1 inhibitors in A1 dependent Mel501 cells Measured in Cell-Based System Using Plate Reader - 2045-09_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
504392Counterscreen of A1 inhibitors in wild-type Mouse Embryonic Fibroblasts Measured in Cell-Based System Using Plate Reader - 2045-04_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatory same project related to Summary assay
504403Counterscreen of A1 inhibitors in Bax/Bak -/- Mouse Embryonic Fibroblasts Measured in Cell-Based System Using Plate Reader - 2045-02_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
504405Counterscreen of A1 inhibitors in wild-type Mouse Embryonic Fibroblasts Measured in Cell-Based System Using Plate Reader - 2045-04_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
504409Secondary assay of A1 inhibitors in Mouse Embryonic Fibroblasts with alternate A1 construct Measured in Cell-Based System Using Plate Reader - 2045-05_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
504412Secondary screen of A1 inhibitors measured by cytochrome c ELISA in CHL-1 cells expressing A1-2A-BIM Measured in Biochemical System Using Plate Reader - 2045-19_Inhibitor_SinglePoint_DryPowder_ActivityOther same project related to Summary assay
504413Inhibitors of A1-dependent Mouse Embryonic Fibroblasts Measured in Cell-Based System Using Plate Reader - 2045-01_Inhibitor_Dose_DryPowder_Activity_3Confirmatory same project related to Summary assay
504415Counterscreen of A1 inhibitors in HMC1-8 Mcl1 dependent cells, viability readout Measured in Cell-Based System Using Plate Reader - 2045-12_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
Description:
Keywords: apoptosis, BH3 domain, Bcl2-A1, BIM, caspase, cancer, cytochrome c

Primary Collaborator: Todd Golub, Broad Institute, golub@broadinstitute.org

Assay Overview:
The fate of cell survival versus apoptosis is determined by the balance of anti and pro-apoptotic proteins. Expression of activator BH3-only proteins, such as BIM or tBID, leads to downstream caspase activation and apoptosis. A1 can functionally bind to and sequester BIM or tBID. An A1 inhibitor causes the release of A1-bound BIM, which activates BAX/BAK, and leads to caspase activation and apoptosis characterized by the release of cytochrome c from the mitochondria. Apoptosis can be confirmed as the mechanism of cell death by measuring the amount of cytochrome c released into the supernatant in this experiment. Levels of cytochrome C are determined using an Enzyme-Linked Immunosorbant Assay (ELISA) with an antibody specific for the protein.

Expected Outcome: Compounds that cause caspase activation will show an increase in absorbance in the ELISA measuring cytochrome c in the supernatant. In this cell line, which does not express A1, compounds that are specific for an A1-dependent apoptotic pathway will not show highly elevated levels of cytochrome c release.
Protocol
CHL-1 cells were grown on 150 mm dishes, then washed with 1X PBS, and 3 ml of trypsin were added. After cells were dislodged, 10ml cold media were added to collect the cells. The cells were centrifuged at 1000 rpm at 4 degrees C for 4 minutes. The cell pellets were washed with cold PBS once, and resuspended in 1X AT buffer (300 mM trehalose, 10 mM HEPES-KOH pH 7.7, 10 mM KCl, 1 mM EGTA, 1 mM EDTA and 0.1% BSA), and homogenized with a Potter Elvehjem homogenizer for 30-40 strokes at 1,600 rpm on ice. After centrifuge at 600g for 10 minutes at 4 degrees C, the supernatant was centrifuged at 7000g for 10 minutes at 4 degrees C. The mitochondria pellets were carefully dislodged and re-suspended in AT buffer with 80mM KCl. The mitochondria were then aliquoted in 96 well assay plates, and incubated with appropriate compounds for 30 minutes at 30oC, then centrifuged at 8000 g for 10 minutes. Negative controls were treated with an equivalent amount of DMSO to compound treatment. Positive controls were treated with equivalent amounts of DMSO + 15 ng recombinant pro-apoptotic protein tBid. Supernatant was collected, diluted at appropriate fold into the 96 well assay kit, and detected with a human cyt c ELISA kit (R&D # SCTC0). Absorbance was read at 450nm in an M5e plate reader (Spectramax), normalized at 540 nm.
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC; n=2) and positive control wells (PC; n=2) were included on the plate.

EXPECTED OUTCOME: Active compounds result in increasing readout signal.

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Stimulators Minus Neutral Controls' 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.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the normalized percent activity of the 50 uM sample, rounded to the nearest integer. The compound(s) were also tested at 10 uM; the percent activity at that concentration is included here for reference.

PUBCHEM_ACTIVITY_OUTCOME:
Activity Outcome = 2 (active) when the PUBCHEM_ACTIVITY_SCORE was at least 10.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1Activity_at_50uM_(%) (50μM**)The percent activity at the indicated concentration.Float%
2Activity_at_10uM_(%) (10μM**)The percent activity at the indicated concentration.Float%

** Test Concentration.
Additional Information
Grant Number: 1 R03 DA028853-01

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