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BioAssay: AID 624069

Vero 76 Cytoxicity Assay for VEEV Compounds (5)

Introduction: 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(10)
 
 
Inactive(10)
 
 
 Tested Substances
 Tested Substances
All(10)
 
 
Inactive(10)
 
 
 Related BioAssays
 Related BioAssays
AID: 624069
Data Source: Southern Research Specialized Biocontainment Screening Center (VEE_VERO_05)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2012-04-11
Hold-until Date: 2013-04-10
Modify Date: 2013-08-29

Data Table ( Complete ):           All
Tested Compounds:
Depositor Specified Assays
Show more
AIDNameTypeComment
588727A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83confirmatoryPrimary and Confirmatory Screen (TC-83)
602241A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (2)confirmatoryConfirmatory Screen (TC-83)
602242A Cell-Based Counter Screen testing Compounds that Inhibit VEEV TC-83, in strain KU V3526confirmatoryConfirmatory Screen (v3586)
602455A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (3)confirmatoryConfirmatory Screen (TC-83)
602484A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, Trinidad Donkey (TrD) (1)confirmatoryconfirmatory Trinidad Donkey
588719Vero 76 Cytoxicity Assay for VEEV CompoundsconfirmatoryCytotoxicity (Vero76) Counter Screen
602439Vero 76 Cytoxicity Assay for VEEV Compounds (3)confirmatoryCytotoxicity (Vero76) Counter Screen
602240Vero 76 Cytoxicity Assay for VEEV Compounds (2)confirmatoryCytotoxicity (Vero76) Counter Screen
623936Vero 76 Cytoxicity Assay for VEEV Compounds (4)confirmatoryCytotoxicity (Vero 76) counter screen
602483HEp2 Cytoxicity Assay for VEEV Compounds (1)confirmatoryCytotoxicity (Hep-2) counter screen
602307Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (TC-83 strain)otherVirus titer reduction assay TC-83
602411Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (2) Trinidad donkey strainotherVirus titer reduction assay (WT-Trinidad Donkey)
602470A Counter Screen of Venezuelan Equine Encephalitis Virus (VEEV) Inhibitors in a Cell-Based Anti-Respiratory Syncytial Virus (RSV) AssayconfirmatoryRespiratory Syncytial Virus (RSV) Counter Screen
623935A Cell-Based Secondary Assay for Compounds that Inhibit VEEV, TC-83 and other Alphaviruses (Chikungunya virus)confirmatoryChikungunya Virus Counter Screen
588723A Cell-based HTS to discover molecules that inhibit VEEV, encephalitic alphavirus - Project SummarysummarySummary AID
651874A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, Trinidad Donkey (TrD) (2)confirmatory
651932A Counter Screen of Venezuelan Equine Encephalitis Virus (VEEV) Inhibitors in a Cell-Based Anti-Respiratory Syncytial Virus (RSV) Assay (2)confirmatory
651884A Cell-Based Counter Screen testing Compounds that Inhibit VEEV TC-83, in strain KU V3526 (2)confirmatory
651934A Cell-Based Secondary Assay for Compounds that Inhibit VEEV, TC-83 and other Alphaviruses (Chikungunya virus) (3)confirmatory
651578Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (TC-83 strain) (4)other
651883Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (strain Trinidad donkey) (03)other
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
Description:
Vero 76 Cytoxicity Assay for VEEV Compounds
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

Introduction: 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). Compounds were considered "Active" (showing toxicity) if they decreased cell viability below 70%. For "Active" compounds, 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) and CC50 values were calculated. 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
BAO: version: 1.4b1090

BAO: bioassay specification: assay stage: Secondary:counterscreening

BAO: bioassay specification: assay biosafety level: BSL2

BAO: assay format: cell-based format

BAO: bioassay specification: assay measurement type: endpoint assay

BAO: format detail: reagent: inducer: none

BAO: meta target: molecular target: protein target: enzyme: transferase: kinase

BAO: meta target: biological process target: cell death

BAO: meta target detail: binding reporter specification: interaction: protein:small molecule

BAO: assay design: viability reporter:atp content

BAO: detection technology: luminescence: chemiluminescence

Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1CC50 *FloatμM
2% Cell Viability @ 25 uM (25μM**)Float%
3% Cell Viability @ 12.5 uM (12.5μM**)Float%
4% Cell Viability @ 6.25 uM (6.25μM**)Float%
5% Cell Viability @ 3.13 uM (3.13μM**)Float%
6% Cell Viability @ 1.56 uM (1.56μM**)Float%
7% Cell Viability @ 0.78 uM (0.78μM**)Float%
8% Cell Viability @ 0.39 uM (0.39μM**)Float%
9% Cell Viability @ 0.2 uM (0.2μM**)Float%

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

Data Table (Concise)
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