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BioAssay: AID 651640

DENV2 CPE-Based HTS Measured in Cell-Based and Microorganism Combination System Using Plate Reader - 2149-01_Other_SinglePoint_HTS_Activity

Assay Overview: The cytopathic effect (CPE) assay has been successfully adapted for studying different virus with aims of identifying novel antiviral compounds. The goal of this assay is to identify inhibitors of Dengue virus, by using a CPE assay. BHK-21 cells were treated with compounds and infected with Dengue virus 2 in 1536-well plates. After a 5-day incubation, CellTiter-Glo reagent (Promega) was added to quantitate cell viability. ..more
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 Tested Compounds
 Tested Compounds
All(343302)
 
 
Active(5946)
 
 
Inactive(321634)
 
 
Inconclusive(16325)
 
 
 Tested Substances
 Tested Substances
All(347136)
 
 
Active(5952)
 
 
Inactive(324845)
 
 
Inconclusive(16339)
 
 
 Related BioAssays
 Related BioAssays
AID: 651640
Data Source: Broad Institute (2149-01_Other_SinglePoint_HTS_Activity)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-10-12

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 5946
Related Experiments
AIDNameTypeComment
651637Broad Institute A CPE-Based HTS Assay for Antiviral Drug Screening Against Dengue Virus Inhibitor Probe ProjectSummarydepositor-specified cross reference
540333A CPE Based HTS Assay for Antiviral Drug Screening Against Dengue VirusScreeningsame project related to Summary assay
687020Mammalian cell toxicity in Vero cells Measured in Cell-Based System Using Plate Reader - 2149-04_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
687028Mammalian cell toxicity in A549 cells Measured in Cell-Based System Using Plate Reader - 2149-02_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
687030Mammalian cell toxicity in BHK-21 cells Measured in Cell-Based System Using Plate Reader - 2149-03_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
687031DENV2 CPE-Based HTS Measured in Cell-Based and Microorganism Combination System Using Plate Reader - 2149-01_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
Description:
Keywords: Dengue Virus 2, Flaviviridae, Cytopathic Effect, BHK-21 Cells, Antivirals

Assay Overview: The cytopathic effect (CPE) assay has been successfully adapted for studying different virus with aims of identifying novel antiviral compounds. The goal of this assay is to identify inhibitors of Dengue virus, by using a CPE assay. BHK-21 cells were treated with compounds and infected with Dengue virus 2 in 1536-well plates. After a 5-day incubation, CellTiter-Glo reagent (Promega) was added to quantitate cell viability.

Expected Outcome: Compounds that register an increase in ATP levels due to increased cell viability compared to positive (uninfected cells) and neutral (DMSO) controls will be considered positives in this assay.
Protocol
Dengue Virus 2 Cytopathic Effect Assay
Virus: Dengue Virus 2 (ATCC VR-1584, stocks prepared by Southern Research Institute, Birmingham, AL, at a titer of 10;5.58 TCDI50/ml)
Cell Line: BHK-21 (ATCC CCL-10)
Materials:
Propagation media: MEM (Cellgro, 15-010-CM), 10% FBS, 1%PSG
Infection media: MEM (Cellgro, 15-010-CM), 2% FBS, 1%PSG
CellTiter-Glo: CellTiter-Glo Custom reagent (Promega)
Procedures:
1.1536-well assay ready plates (ARPs) were previously prepared by acoustic transfer (Echo555, LabCyte) with the addition of 5nl of compound to each well. One day before the initiation of the assay, the evaporation barriers of the ARPs were filled with MEM + 1% PSG.
2.Six to seven days prior to starting the assay, BHK-21 cells were thawed into Propagation media and grown for 3 days. Cells were then split so as to be close to full confluency after 3-4 days.
3.BHK-21 cells were harvested, counted, and resuspended to 75,000 cells/ml in Infection media. Cells were plated into the ARPs at 375 cells/well in 5ul.
4.200nl of Dengue virus to a final concentration of 1:25 was added to every well except the positive control wells. The final concentration of compound was 10uM in each well. Plates were incubated at 37 degrees C, 5%CO2, 95% relative humidity for 5 days.
5.After 5 days of incubation, plates were removed from the incubator to cool to room temperature for 10 minutes. Following the cooling, 1.5ul of CellTiter-Glo was added and the plates were set to incubate for 10 minutes at room temperature. At the conclusion of the incubation, plates were read with a plate reader for luminescence (0.1 sec. standard luminescence).
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every 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 mean of the normalized sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 20.
PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 100.
PUBCHEM_ACTIVITY_OUTCOME:
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T
Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T
Samples passing AT only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T
Samples passing PAR_T only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based/Organism-based
Assay Cell Type: BHK-21
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1REPRODUCIBILITY_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
2PCT_ACTIVE_REPLICATESThe percentage of replicates which pass the activity threshold.Float
3REPLICATE_A_ACTIVITY_SCORE_9.99uM_(%) (9.99μM**)The calculated activity for the indicated sampleFloat%
4REPLICATE_B_ACTIVITY_SCORE_9.99uM_(%) (9.99μM**)The calculated activity for the indicated sampleFloat%

** Test Concentration.
Additional Information
Grant Number: 1 RO3 MH090816-01A1

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