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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 ):           View All Data
Tested Compounds:
Related Experiments
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AIDNameTypeComment
588719Vero 76 Cytoxicity Assay for VEEV CompoundsConfirmatorydepositor-specified cross reference: Cytotoxicity (Vero76) Counter Screen
588723A Cell-based HTS to discover molecules that inhibit VEEV, encephalitic alphavirus - Project SummarySummarydepositor-specified cross reference: Summary AID
588727A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83Confirmatorydepositor-specified cross reference: Primary and Confirmatory Screen (TC-83)
602240Vero 76 Cytoxicity Assay for VEEV Compounds (2)Confirmatorydepositor-specified cross reference: Cytotoxicity (Vero76) Counter Screen
602241A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (2)Confirmatorydepositor-specified cross reference: Confirmatory Screen (TC-83)
602242A Cell-Based Counter Screen testing Compounds that Inhibit VEEV TC-83, in strain KU V3526Confirmatorydepositor-specified cross reference: Confirmatory Screen (v3586)
602307Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (TC-83 strain)Otherdepositor-specified cross reference: Virus titer reduction assay TC-83
602411Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (2) Trinidad donkey strainOtherdepositor-specified cross reference: Virus titer reduction assay (WT-Trinidad Donkey)
602439Vero 76 Cytoxicity Assay for VEEV Compounds (3)Confirmatorydepositor-specified cross reference: Cytotoxicity (Vero76) Counter Screen
602455A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (3)Confirmatorydepositor-specified cross reference: Confirmatory Screen (TC-83)
602470A Counter Screen of Venezuelan Equine Encephalitis Virus (VEEV) Inhibitors in a Cell-Based Anti-Respiratory Syncytial Virus (RSV) AssayConfirmatorydepositor-specified cross reference: Respiratory Syncytial Virus (RSV) Counter Screen
602483HEp2 Cytoxicity Assay for VEEV Compounds (1)Confirmatorydepositor-specified cross reference: Cytotoxicity (Hep-2) counter screen
602484A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, Trinidad Donkey (TrD) (1)Confirmatorydepositor-specified cross reference: confirmatory Trinidad Donkey
623935A Cell-Based Secondary Assay for Compounds that Inhibit VEEV, TC-83 and other Alphaviruses (Chikungunya virus)Confirmatorydepositor-specified cross reference: Chikungunya Virus Counter Screen
623936Vero 76 Cytoxicity Assay for VEEV Compounds (4)Confirmatorydepositor-specified cross reference: Cytotoxicity (Vero 76) counter screen
651578Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (TC-83 strain) (4)Otherdepositor-specified cross reference
651874A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, Trinidad Donkey (TrD) (2)Confirmatorydepositor-specified cross reference
651883Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (strain Trinidad donkey) (03)Otherdepositor-specified cross reference
651884A Cell-Based Counter Screen testing Compounds that Inhibit VEEV TC-83, in strain KU V3526 (2)Confirmatorydepositor-specified cross reference
651886Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (TC-83 strain) (5)Otherdepositor-specified cross reference
651917A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (8)Confirmatorydepositor-specified cross reference
651930Vero 76 Cytoxicity Assay for VEEV Compounds (9)Confirmatorydepositor-specified cross reference
651932A Counter Screen of Venezuelan Equine Encephalitis Virus (VEEV) Inhibitors in a Cell-Based Anti-Respiratory Syncytial Virus (RSV) Assay (2)Confirmatorydepositor-specified cross reference
651934A Cell-Based Secondary Assay for Compounds that Inhibit VEEV, TC-83 and other Alphaviruses (Chikungunya virus) (3)Confirmatorydepositor-specified cross reference
624063A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV TC-83 (4)Confirmatorysame project related to Summary assay
624284A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (5)Confirmatorysame project related to Summary assay
624286Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (TC-83 strain) (3)Othersame project related to Summary assay
624295Vero 76 Cytoxicity Assay for VEEV Compounds (6)Confirmatorysame project related to Summary assay
624449A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (6)Confirmatorysame project related to Summary assay
624450Vero 76 Cytoxicity Assay for VEEV Compounds (7)Confirmatorysame project related to Summary assay
651734A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (7)Confirmatorysame project related to Summary assay
651735Vero 76 Cytoxicity Assay for VEEV Compounds (8)Confirmatorysame project related to Summary assay
651738A Cell-Based Secondary Assay for Compounds that Inhibit VEEV, TC-83 and other Alphaviruses (Chikungunya virus) (2)Confirmatorysame project related to Summary assay
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 - additional comments and annotations
From BioAssay Depositor:
BAO: assay design: viability reporter:atp content
BAO: assay format: cell-based format
BAO: bioassay specification: assay biosafety level: BSL2
BAO: bioassay specification: assay measurement type: endpoint assay
BAO: bioassay specification: assay stage: Secondary:counterscreening
BAO: detection technology: luminescence: chemiluminescence
BAO: format detail: reagent: inducer: none
BAO: meta target detail: binding reporter specification: interaction: protein:small molecule
BAO: meta target: biological process target: cell death
BAO: meta target: molecular target: protein target: enzyme: transferase: kinase
BAO: version: 1.4b1090
From PubChem:
Assay Type: Toxicity
Assay Cell Type: VERO 76
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)
Data Table ( Complete ):     View All Data
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