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BioAssay: AID 588719

Vero 76 Cytoxicity Assay for VEEV Compounds

This functional assay was developed for detection of compounds inhibiting Vero 76 cells viability as a secondary screen to the VEEV Inhibition screen. ..more
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 Tested Compounds
 Tested Compounds
All(1481)
 
 
Active(917)
 
 
Inactive(564)
 
 
 Tested Substances
 Tested Substances
All(1481)
 
 
Active(917)
 
 
Inactive(564)
 
 
 Related BioAssays
 Related BioAssays
AID: 588719
Data Source: Southern Research Specialized Biocontainment Screening Center (VEE_VERO_01)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2011-10-27
Modify Date: 2011-10-30

Data Table ( Complete ):           Active    All
BioActive Compounds: 917
Depositor Specified Assays
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AIDNameTypeComment
588723A Cell-based HTS to discover molecules that inhibit VEEV, encephalitic alphavirus - Project Summarysummary
602240Vero 76 Cytoxicity Assay for VEEV Compounds (2)confirmatory
602241A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (2)confirmatory
602242A Cell-Based Counter Screen testing Compounds that Inhibit VEEV TC-83, in strain KU V3526confirmatory
602307Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (TC-83 strain)other
602439Vero 76 Cytoxicity Assay for VEEV Compounds (3)confirmatory
602455A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (3)confirmatory
602470A Counter Screen of Venezuelan Equine Encephalitis Virus (VEEV) Inhibitors in a Cell-Based Anti-Respiratory Syncytial Virus (RSV) Assayconfirmatory
602483HEp2 Cytoxicity Assay for VEEV Compounds (1)confirmatory
602484A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, Trinidad Donkey (TrD) (1)confirmatory
623935A Cell-Based Secondary Assay for Compounds that Inhibit VEEV, TC-83 and other Alphaviruses (Chikungunya virus)confirmatory
623936Vero 76 Cytoxicity Assay for VEEV Compounds (4)confirmatory
624063A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV TC-83 (4)confirmatory
624069Vero 76 Cytoxicity Assay for VEEV Compounds (5)confirmatory
624284A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (5)confirmatory
624286Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (TC-83 strain) (3)other
624295Vero 76 Cytoxicity Assay for VEEV Compounds (6)confirmatory
624449A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (6)confirmatory
624450Vero 76 Cytoxicity Assay for VEEV Compounds (7)confirmatory
651578Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (TC-83 strain) (4)other
651734A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (7)confirmatory
651735Vero 76 Cytoxicity Assay for VEEV Compounds (8)confirmatory
651738A Cell-Based Secondary Assay for Compounds that Inhibit VEEV, TC-83 and other Alphaviruses (Chikungunya virus) (2)confirmatory
651874A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, Trinidad Donkey (TrD) (2)confirmatory
651883Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (strain Trinidad donkey) (03)other
651884A Cell-Based Counter Screen testing Compounds that Inhibit VEEV TC-83, in strain KU V3526 (2)confirmatory
651886Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (TC-83 strain) (5)other
651917A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (8)confirmatory
651930Vero 76 Cytoxicity Assay for VEEV Compounds (9)confirmatory
651932A Counter Screen of Venezuelan Equine Encephalitis Virus (VEEV) Inhibitors in a Cell-Based Anti-Respiratory Syncytial Virus (RSV) Assay (2)confirmatory
651934A Cell-Based Secondary Assay for Compounds that Inhibit VEEV, TC-83 and other Alphaviruses (Chikungunya virus) (3)confirmatory
Description:
Southern Research's Specialized Biocontainment Screening Center (SRSBSC)
Southern Research Institute (Birmingham, Alabama)
NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Assay Provider: Dong Hoon Chung, University of Louisville
Award: 1 R03 MH087448-01A1

This functional assay was developed for detection of compounds inhibiting Vero 76 cells viability as a secondary screen to the VEEV Inhibition screen.

In this assay, we treated Vero 76 cells with compounds selected as hits in the VEEV Inhibition assay for 72 hours over a 10 point 2-fold dilution series, ranging from 25 uM to 0.05 uM. Following 72 hours of treatment, relative viable cell number was determined using Cell Titer Glo from Promega. Each plate contained 64 replicates of vehicle treated cells which served as negative controls.
Protocol
Cell Culture: Vero 76 cells obtained from ATCC (CRL-1587) were cultured and maintained in MEM-E (Invitrogen, 10370-088) with 10% Hi-FBS (Invitrogen 16000), 1% Penicillin/Streptomycin/L-glutamine (Invitrogen 10378-024) and 1% HEPES (Invitrogen 15630-080). The cells are maintained at 37C, 5.0% CO2 to 100% confluence being passaged 1:4 every 3-4 days. For cell plating, cells were detached from flask bottom by using Trypsin-EDTA solution and then re-suspended in a growth media. Cells were passaged no more than ten times after being thawed.

Compound Dosing/Plating: Carrier control / compounds were diluted in complete growth medium to prepare a 6X concentrated dosing solution which was dispensed into 384-well black clear-bottom tissue culture treated plates (5 uL volume).

Cell Plating: Twenty-five uL of complete growth medium containing 3000 cells were dispensed per well. Plates were incubated at 37 C, 5% CO2 for 72h prior to endpoint detection.

Endpoint/Detection: At the end of the treatment period, assay plates were removed from the incubator and equilibrated to room temperature for 10 min. Thirty uL of Cell Titer Glo reagent was added and plates were incubated for an additional 10 min in the dark. At the end of the incubation, assay plates were analyzed using a PerkinElmer Envision microplate reader in luminescence mode with an integration time of 0.1 s.

Data Analysis: Sixty-four control wells containing cells treated with DMSO vehicle and were included on each assay plate. Compound data was normalized and reported as % viability which was calculated using the following formula: % viability = 100*(Cmpd Lum-Med background)/(Med Cell Ctrl - Med background). The normalized % viability was plotted against the tested concentrations. The CC50 values were calculated using XLfit formula 205, a 4 parameter Levenburg-Marquardt algorithm with maximum and minimum limits set at 100 and 0, respectively and allowing extrapolation to identify weakly active compounds.
Comment
Outcome: Compounds that showed <70% cell viability for at least one concentration were defined as "Active" (toxic). If the % viability at all doses was >70%, the compound was defined as "Inactive" (non-toxic).

The following tiered system has been implemented at Southern Research Institute for use with the PubChem Score: Compounds in the primary screen are scored on a scale of 0-40 based on % activity; a score of 40 corresponds to 100% activity. In the confirmatory dose response screen of primary screen hits, active compounds are scored on a scale of 41-80 based on IC50 result while compounds where activity was not confirmed are given the score 0. Confirmatory dose response and secondary screens of purified and/or resynthesized compounds, indicating the highest degree of confidence) are scored on a scale of 81-100 based on IC50 result. Inactive compounds are given the score 0.
Categorized Comment
Phenotypic Screen: Yes

Multiplexing: Yes

BSL: BSL2

Screening Concentration
Screening Concentration Range Max: 25

Screening Concentration Range Min: 0.049

Assay Format: Cell-based

Assay Type: Viability/Toxicity

Assay Method: End-point

Assay Detection: Bio-luminescence

Used for Hit Validation?: Yes

Used during SAR?: Yes

Secondary Assay Sub-type: Counter-screen Assay

Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1CC50 Modifier String
2CC50 *FloatμM
3StDev CC50 Float
4CC50 Hill Slope Float
5CC50 NormChi2 Float
6% Viability @ 25 uM (25μM**)Float%
7% Viability @ 12.5 uM (12.5μM**)Float%
8% Viability @ 6.25 uM (6.25μM**)Float%
9% Viability @ 3.13 uM (3.13μM**)Float%
10% Viability @ 1.56 uM (1.56μM**)Float%
11% Viability @ 0.78 uM (0.78μM**)Float%
12% Viability @ 0.39 uM (0.39μM**)Float%
13% Viability @ 0.19 uM (0.19μM**)Float%
14% Viability @ 0.09 uM (0.09μM**)Float%
15% Viability @ 0.05 uM (0.05μM**)Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH087448-01A1

Data Table (Concise)
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