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BioAssay: AID 2509

Fluorescence Cell-Based Screen to Identify Inhibitors of Phorbol Myristate Acetate-Mediated P-Selectin Induction on Platelets

Assay Overview: Cell-based assay for inhibition of PMA (phorbol myristate acetate)-induced P-selectin surface expression. Washed platelets obtained from individual donors were treated with compounds found to show less than 50% inhibition in an SFLLRN-induced FITC phalloidin assay. Platelet samples were treated with 30 uM compound. Following compound addition, platelets were stimulated with 5 more ..
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 Tested Compounds
 Tested Compounds
All(2)
 
 
Inactive(2)
 
 
 Tested Substances
 Tested Substances
All(2)
 
 
Inactive(2)
 
 
 Related BioAssays
 Related BioAssays
AID: 2509
Data Source: Broad Institute (2016-07_INHIBITORS_SINGLE-POINT_MLPCN-CHERRYPICK)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2010-03-11
Hold-until Date: 2010-09-30
Modify Date: 2010-09-30

Data Table ( Complete ):           View All Data
Tested Compounds:
Related Experiments
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AIDNameTypeProbeComment
1663MLPCN Platelet Activation -Dense Granule ReleaseScreening depositor-specified cross reference: Primary HTS
1678Broad Institute MLPCN Platelet ActivationSummary3 depositor-specified cross reference: Project Summary
1889Luminescence Cell-Based Dose Confirmation HTS to Identify Inhibitors of Platelet Dense Granule ReleaseConfirmatory same project related to Summary assay
1891Luminescence Biochemical Dose Response HTS to Identify Inhibitors of LuciferaseConfirmatory same project related to Summary assay
2398Luminescence Cell-Based Dose Response Followup to Identify Inhibitors of Platelet Dense Granule ReleaseConfirmatory same project related to Summary assay
2511Fluorescence Cell-Based Dose Response to Confirm Inhibitors of Thrombin Receptor-Activating Peptide SFLLRN-Mediated P-Selectin Induction on PlateletsConfirmatory same project related to Summary assay
2518Fluorescence Cell-Based Dose Response to Identify Inhibitors of Thrombin Receptor-Activating Peptide SFLLRN-Mediated P-Selectin Induction on Platelets.Confirmatory same project related to Summary assay
2519Luminescence Cell-Based Dose Response Followup to Identify Inhibitors of Platelet Dense Granule Release.Confirmatory same project related to Summary assay
2522Fluorescence Cell-Based Screen to Identify Inhibitors of Calcium Ionophore-Mediated P-Selectin Induction on PlateletsScreening same project related to Summary assay
2527Fluorescence Cell-Based Screen to Confirm Inhibitors of Calcium Ionophore-Mediated P-Selectin Induction on PlateletsScreening same project related to Summary assay
2529Fluorescence Cell-Based Screen to Confirm Inhibitors of Phorbol Myristate Acetate-Mediated P-Selectin Induction on PlateletsScreening same project related to Summary assay
2645ELISA Cell-Based Screen to Identify Inducers of cAMP in PlateletsScreening same project related to Summary assay
2646ELISA Cell-Based Screen to Identify an Inducer of cAMP in PlateletsScreening same project related to Summary assay
2655Fluorescence Cell-Based Screen to Identify an Inhibitor of Thrombin Receptor-Activating Peptide SFLLRN-Mediated Actin Polymerization in PlateletsScreening same project related to Summary assay
2656Radioactive Cell-Based Screen to Identify Inhibitors of Thrombin Receptor-Activating Peptide SFLLRN-Mediated Dense Granule Release by PlateletsScreening same project related to Summary assay
2657Fluorescence Cell-Based Screen to Identify Inhibitors of Thrombin Receptor-Activating Peptide SFLLRN-Mediated Actin Polymerization in PlateletsScreening same project related to Summary assay
493031SFLLRN-induced P-Selectin Platelet Surface Expression Measured in Cell-Based System Using Flow Cytometry - 2016-03_Inhibitor_SinglePoint_DryPowder_ActivityOther same project related to Summary assay
493068SFLLRN-induced P-Selectin Platelet Surface Expression Measured in Cell-Based System Using Flow Cytometry - 2016-03_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
493100Platelet granule secretion Measured in Cell-Based System Using Plate Reader - 2016-01_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
Description:
Keywords: Platelet, activation, P-selectin, PMA, Protein kinase C

Assay Overview: Cell-based assay for inhibition of PMA (phorbol myristate acetate)-induced P-selectin surface expression. Washed platelets obtained from individual donors were treated with compounds found to show less than 50% inhibition in an SFLLRN-induced FITC phalloidin assay. Platelet samples were treated with 30 uM compound. Following compound addition, platelets were stimulated with 5 nM PMA. After a 15-minute incubation, phycoerythrin-labeled anti-P-selectin antibody (BD Biosciences) was added for a 20-minute incubation. The samples were analyzed by flow cytometry to determine P-selectin expression on the surface of the platelets as a response to activation. Geometric mean values were collected for each sample.

Expected Outcome: This assay serves as a specificity assay, along with an analogous assay using Ca2+-ionophore-induced activation of platelets, to determine selectivity towards G-protein coupled receptors (GPCR). Compounds that do not inhibit surface expression of P-selectin from PMA-induced and Ca2+-ionophore-induced activation are potential GPCR-selective inhibitors.

Primary Collaborators(and laboratory where this assay was performed):
Robert Flaumenhaft, Beth Israel Deaconess Medical Center,rflaumen@bidmc.harvard.edu,617-754-1204
John Thomas,NHLBI,ThomasJ@nhlbi.nih.gov
Protocol
1. Platelet samples (10 ul) were incubated with 30 uM compound.

2. 20 minutes following compound addition, platelets were stimulated with 5 nM PMA.

3. After a 15-minute incubation, phycoerythrin-labeled anti-P-selectin antibody (5 ul; BD Biosciences) was added. The samples were agitated gently.

4. After a 20-minute incubation, 500 ul of FACS buffer (BD Biosciences) was added to each of the samples.

5. Samples were then analyzed by flow cytometry to determine P-selectin expression on the surface of the platelets as a response to activation.

6. Geometric mean values were collected for each sample.
Comment
Data analysis:
The neutral control was: DMSO only, with no compound.
Compounds were tested at 30uM.

Percent inhibition of induced P-Selectin expression was calculated as:
100 - (100 * (Mean Fluorescence at 30uM/ Mean Fluorescence at DMSO))
The percent inhibition is reported for two replicates in each run.

PUBCHEM_ACTIVITY_SCORE
The average Percent Inhibition value at the tested concentration 30uM, calculated across all runs.

PUBCHEM_ACTIVITY_OUTCOME
Activity_Outcome = 1 (inactive)
score <= 50 in all runs

Activity_Outcome = 2 (active)
score > 50 in all runs

Activity_Outcome = 3 (inconclusive)
score > 50 in one or more runs and score <= 50 in one or more runs

Compounds were retested at least once. These retests were analyzed as independent tests; therefore each test generated a separate set of replicate data, including a score (average percent inhibition across replicates) and an outcome (2 (active) for score > 50).
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1outcome_Run1The activity outcome for Run1Integer
2score_Run1The activity ranking score for Run1Integer
3REPLICATE_A_ACTIVITY_SCORE_Run1The calculated activity score for the sample A, a NULL here denotes data was not used or was not producedFloat%
4REPLICATE_B_ACTIVITY_SCORE_Run1The calculated activity score for the sample A, a NULL here denotes data was not used or was not producedFloat%
5outcome_Run2the fitted activity level at infinite concentrationInteger
6score_Run2the slope at EC50Integer
7REPLICATE_A_ACTIVITY_SCORE_Run2The calculated activity score for the sample A, a NULL here denotes data was not used or was not producedFloat%
8REPLICATE_B_ACTIVITY_SCORE_Run2The calculated activity score for the sample A, a NULL here denotes data was not used or was not producedFloat%
Additional Information
Grant Number: 1R03DA026209-01

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