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BioAssay: AID 651930

Vero 76 Cytoxicity Assay for VEEV Compounds (9)

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(43)
 
 
Active(1)
 
 
Inactive(42)
 
 
 Tested Substances
 Tested Substances
All(44)
 
 
Active(1)
 
 
Inactive(43)
 
 
 Related BioAssays
 Related BioAssays
AID: 651930
Data Source: Southern Research Specialized Biocontainment Screening Center (VEE_VERO_09)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-12-12
Hold-until Date: 2013-12-01
Modify Date: 2013-12-02

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compound: 1
Related Experiments
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AIDNameTypeComment
588719Vero 76 Cytoxicity Assay for VEEV CompoundsConfirmatorydepositor-specified cross reference: Cytotoxicity counter screen (Vero 76)
588723A Cell-based HTS to discover molecules that inhibit VEEV, encephalitic alphavirus - Project SummarySummarydepositor-specified cross reference
588727A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83Confirmatorydepositor-specified cross reference: Primary and Confirmatory Assays
602240Vero 76 Cytoxicity Assay for VEEV Compounds (2)Confirmatorydepositor-specified cross reference: Cytotoxicity counter screen (Vero 76)
602241A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (2)Confirmatorydepositor-specified cross reference: Confirmatory (TC-83)
602242A Cell-Based Counter Screen testing Compounds that Inhibit VEEV TC-83, in strain KU V3526Confirmatorydepositor-specified cross reference: Counter (v3586)
602307Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (TC-83 strain)Otherdepositor-specified cross reference: Virus Titer Reduction (TC-83)
602411Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (2) Trinidad donkey strainOtherdepositor-specified cross reference: Virus Titer Reduction (Trinidad Donkey)
602439Vero 76 Cytoxicity Assay for VEEV Compounds (3)Confirmatorydepositor-specified cross reference: Cytotoxicity counter screen (Vero 76)
602455A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (3)Confirmatorydepositor-specified cross reference: Confirmatory (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: RSV
602483HEp2 Cytoxicity Assay for VEEV Compounds (1)Confirmatorydepositor-specified cross reference: Cytotoxicity counter screen (Hep-2)
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: CHIKV
623936Vero 76 Cytoxicity Assay for VEEV Compounds (4)Confirmatorydepositor-specified cross reference: Cytotoxicity counter screen (Vero 76)
624063A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV TC-83 (4)Confirmatorydepositor-specified cross reference: Confirmatory (TC-83)
624069Vero 76 Cytoxicity Assay for VEEV Compounds (5)Confirmatorydepositor-specified cross reference: Cytotoxicity counter screen (Vero 76)
624284A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (5)Confirmatorydepositor-specified cross reference: Confirmatory (TC-83)
624286Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (TC-83 strain) (3)Otherdepositor-specified cross reference: Virus Titer Reduction (TC-83)
624295Vero 76 Cytoxicity Assay for VEEV Compounds (6)Confirmatorydepositor-specified cross reference: Cytotoxicity counter screen (Vero 76)
624449A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (6)Confirmatorydepositor-specified cross reference: Confirmatory (TC-83)
624450Vero 76 Cytoxicity Assay for VEEV Compounds (7)Confirmatorydepositor-specified cross reference: Cytotoxicity counter screen (Vero 76)
651578Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (TC-83 strain) (4)Otherdepositor-specified cross reference: Virus Titer Reduction (TC-83)
651734A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (7)Confirmatorydepositor-specified cross reference: Confirmatory (TC-83)
651738A Cell-Based Secondary Assay for Compounds that Inhibit VEEV, TC-83 and other Alphaviruses (Chikungunya virus) (2)Confirmatorydepositor-specified cross reference: CHIKV
651874A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, Trinidad Donkey (TrD) (2)Confirmatorydepositor-specified cross reference: confirmatory Trinidad Donkey
651883Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (strain Trinidad donkey) (03)Otherdepositor-specified cross reference: VTR (WT-Trinidad Donkey; Hoon)
651884A Cell-Based Counter Screen testing Compounds that Inhibit VEEV TC-83, in strain KU V3526 (2)Confirmatorydepositor-specified cross reference: counter v3586
651886Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (TC-83 strain) (5)Otherdepositor-specified cross reference: Virus Titer Reduction (TC-83)
651735Vero 76 Cytoxicity Assay for VEEV Compounds (8)Confirmatorysame project related to Summary assay
651917A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (8)Confirmatorysame project related to Summary assay
651932A Counter Screen of Venezuelan Equine Encephalitis Virus (VEEV) Inhibitors in a Cell-Based Anti-Respiratory Syncytial Virus (RSV) Assay (2)Confirmatorysame project related to Summary assay
651934A Cell-Based Secondary Assay for Compounds that Inhibit VEEV, TC-83 and other Alphaviruses (Chikungunya virus) (3)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, with various starting concentrations. 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: 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
ScoreThe BioAssay activity ranking scoreInteger
1CC50 ModifierString
2CC50 *FloatμM
3% Viability @ 50 uM Test 1 (50μM**)Float%
4% Viability @ 25 uM Test 1 (25μM**)Float%
5% Viability @ 12.5 uM Test 1 (12.5μM**)Float%
6% Viability @ 6.25 uM Test 1 (6.25μM**)Float%
7% Viability @ 5 uM Test 1 (5μM**)Float%
8% Viability @ 3.125 uM Test 1 (3.125μM**)Float%
9% Viability @ 2.5 uM Test 1 (2.5μM**)Float%
10% Viability @ 1.563 uM Test 1 (1.563μM**)Float%
11% Viability @ 1.25 uM Test 1 (1.25μM**)Float%
12% Viability @ 0.781 uM Test 1 (0.781μM**)Float%
13% Viability @ 0.625 uM Test 1 (0.625μM**)Float%
14% Viability @ 0.391 uM Test 1 (0.391μM**)Float%
15% Viability @ 0.313 uM Test 1 (0.313μM**)Float%
16% Viability @ 0.195 uM Test 1 (0.195μM**)Float%
17% Viability @ 0.156 uM Test 1 (0.156μM**)Float%
18% Viability @ 0.078 uM Test 1 (0.078μM**)Float%
19% Viability @ 0.039 uM Test 1 (0.039μM**)Float%
20% Viability @ 0.02 uM Test 1 (0.02μM**)Float%
21% Viability @ 0.01 uM Test 1 (0.01μM**)Float%
22% Viability @ 0.005 uM Test 1 (0.005μM**)Float%
23% Viability @ 25 uM Test 2 (25μM**)Float%
24% Viability @ 12.5 uM Test 2 (12.5μM**)Float%
25% Viability @ 6.25 uM Test 2 (6.25μM**)Float%
26% Viability @ 5 uM Test 2 (5μM**)Float%
27% Viability @ 3.125 uM Test 2 (3.125μM**)Float%
28% Viability @ 2.5 uM Test 2 (2.5μM**)Float%
29% Viability @ 1.563 uM Test 2 (1.563μM**)Float%
30% Viability @ 1.25 uM Test 2 (1.25μM**)Float%
31% Viability @ 0.781 uM Test 2 (0.781μM**)Float%
32% Viability @ 0.625 uM Test 2 (0.625μM**)Float%
33% Viability @ 0.391 uM Test 2 (0.391μM**)Float%
34% Viability @ 0.313 uM Test 2 (0.313μM**)Float%
35% Viability @ 0.195 uM Test 2 (0.195μM**)Float%
36% Viability @ 0.156 uM Test 2 (0.156μM**)Float%
37% Viability @ 0.078 uM Test 2 (0.078μM**)Float%
38% Viability @ 0.039 uM Test 2 (0.039μM**)Float%
39% Viability @ 0.02 uM Test 2 (0.02μM**)Float%
40% Viability @ 0.01 uM Test 2 (0.01μM**)Float%
41% Viability @ 0.005 uM Test 2 (0.005μM**)Float%
42% Viability @ 0.625 uM Test 3 (0.625μM**)Float%
43% Viability @ 0.313 uM Test 3 (0.313μM**)Float%
44% Viability @ 0.156 uM Test 3 (0.156μM**)Float%
45% Viability @ 0.078 uM Test 3 (0.078μM**)Float%
46% Viability @ 0.039 uM Test 3 (0.039μM**)Float%
47% Viability @ 0.02 uM Test 3 (0.02μM**)Float%
48% Viability @ 0.01 uM Test 3 (0.01μM**)Float%
49% Viability @ 0.005 uM Test 3 (0.005μM**)Float%
50% Viability @ 6.25 uM Test 4 (6.25μM**)Float%
51% Viability @ 3.125 uM Test 4 (3.125μM**)Float%
52% Viability @ 1.563 uM Test 4 (1.563μM**)Float%
53% Viability @ 1.25 uM Test 4 (1.25μM**)Float%
54% Viability @ 0.781 uM Test 4 (0.781μM**)Float%
55% Viability @ 0.625 uM Test 4 (0.625μM**)Float%
56% Viability @ 0.391 uM Test 4 (0.391μM**)Float%
57% Viability @ 0.313 uM Test 4 (0.313μM**)Float%
58VerificationString

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

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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