DENV2 CPE-Based HTS Measured in Cell-Based and Microorganism Combination System Using Plate Reader - 2149-01_Inhibitor_Dose_CherryPick_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: 50
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.Dengue virus was added to each well except the positive control wells, to give a final concentration of 1:25 (primary screen) or 1:17 (retest at dose). The final concentration of compound was 10uM in each well in the primary screen, and ranged in concentration from 20uM to 0.15uM in the retest at dose. 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; n=144) and positive control wells (PC; n=144) were included on every plate.
EXPECTED OUTCOME: Active compounds result in increasing readout signal.
The compounds were assayed in multiple independent instances using an identical protocol; each instance is called a 'test'. For each test, the following analysis was applied:
ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal Max_Concentration.
The raw signals of the plate wells were normalized using the 'Stimulators Minus Neutral Controls' method in Genedata Assay Analyzer (v10.0.2):
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.10.0.2) was applied.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): absACnn, the concentration at which the curve crosses threshold 30.0
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= 30% but <= 70%, or
b) The fit was deemed not valid due to poor fit quality.
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.
Once the data for each test was processed, the test number was appended to all column headers in that test's data set. The individual test results were then aggregated as follows:
1. The final PUBCHEM_ACTIVITY_OUTCOME was set to:
a. '2' (active) when all test outcomes were '2' (active), or
b. '1' (inactive) when all test outcomes were '1' (inactive), or
c. '3' (inconclusive) when the test outcomes were mixed.
2. The final ACTIVE_CONCENTRATION (AC) was set as follows:
a. If the final PUBCHEM_ACTIVITY_OUTCOME = 2, AC was set as the mean of the constituent test active concentrations;
b. If the final PUBCHEM_ACTIVITY_OUTCOME = 1 or 3, AC was left empty.
3. The final PUBCHEM_ACTIVITY_SCORE was calculated based on the aggregated ACTIVE_CONCENTRATION, using the same logic described above for individual test scores.
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
Categorized Comment - additional comments and annotations
Assay Format: Cell-based/Organism-based
Assay Cell Type: BHK-21
Assay Type: Functional
* Activity Concentration. ** Test Concentration.
Data Table (Concise)