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
BioActive Compounds: 5946
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.
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)
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)
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).
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.
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.
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.
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
** Test Concentration.
Data Table (Concise)