Name: a fluorescence-based lymphocyte assay suitable for high-throughput screening of small molecules
Uploaded: 2017-03-10T14:00:00
Description: Watch this Scientific Journal Video about A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules at JoVE.com

Dette arbejde blev støttet af Merck Frosst Start-up midler fra Université de Montréal. Vi vil gerne takke Drs Jean Duchaine og Dominic Salois fra High-throughput platform på Institut for Forskning i Immunologi og kræft for deres diskussion, kommentarer og feedbacks. Moutih Rafei har en Fonds de la Recherche en Santé du Québec Junior 1 Award.

Alle dyr protokoller blev godkendt af Animal Care udvalg Université de Montréal. Mus blev aflivet ved gradvis inhalation af CO2, indtil der ikke vitale tegn blev observeret efterfulgt af cervikal dislokation. Proceduren blev udført af en certificeret person til at sikre, at dyrene blev aflivet på en human måde og i overensstemmelse med anbefalingerne fra det canadiske Rådet om Animal Care. 1. Udarbejdelse af splenocyt Medium og Flow-cytometri Buffer <ol><li> Udfør alle tr...

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Adskillige udlæste metoder er blevet udnyttet til udvikling af følsomme og pålidelige HTS assays. Disse omfatter kolorimetriske, luminescerende eller fluorescerende metoder. Selvom kolorimetriske metoder er enkle at sætte op, de kræver flere tilføjelser af kemikalier, som kan forstyrre eller forstyrre cellerne bliver testet. 23 Hertil kommer, at de ikke tillader dynamisk vurdering af en biologisk reaktion som den farmakologiske virkning vurderes ved en given virkning. Endvidere kan denne fr...

Høj throughput screening (HTS) er en bevist strategi udbredt til identifikation af nye terapeutiske molekyler eller til repositionering af FDA-godkendte lægemidler i nye medicinske indikationer. 1 Indtil videre kan det opnås HTS succes måles ved overfloden af tidligere opdagede lægemidler. For eksempel tyrosinkinaseinhibitoren lapatinib anvendes til behandling af brystkræft, sitagliptin; en dipeptidyl peptidase-4 (DPP-4) -hæmmer anvendes som et anti-hyperglykæmiske lægemiddel og den orale Bcr-Abl tyrosinkinaseinhibitoren dasatinib anvendes til behandling af kronisk myeloid leukæmi repræsenterer få eksempler på en lang liste over godkendte lægemidler oprindeligt opdaget af HTS. 2 Selv om produktiviteten af medicinalindustrien sidst har lidt under manglende i opdagelsen af nye kemiske enheder, kan sandsynligheden for en vellykket lægemiddelforskning forbedres gennem en stigning i antallet af prækliniske candidaTES viser modulerende biologiske / biokemiske egenskaber. Følgelig kunne udviklingen af ​​nye HTS analyser tilpasset til fænotypisk screening giver mulighed for at give vigtige farmakologiske værktøjer til opdagelsen af ​​nye lægemiddelkandidater hits. 3, 4, 5, 6 Endvidere kan HTS nu udføres i et hurtigere tempo på grund af betydelige teknologiske forandringer i de senere år, herunder specialdesignede fleksible robot installationer, nye read-out teknologier og omfattende miniaturisering. 2, 7 Blandt de faktorer, der bidrager til den stigende interesse for brugen af fænotypisk screening (aka fremad farmakologi) er opfattelsen, at fokusere på funktionelle virkninger snarere end forsimplede reduktionistiske antagelser om molekylære mål (mål-baserede screening / biokemiske reaktioner) er mere sandsynligt at sho<html> w klinisk effektivitet. Således fænotypisk screening giver løfte at afdække nye potentielle terapeutiske forbindelser og molekylære veje for øjeblikket uhelbredelige sygdomme. 2 For at kunne identificere inhibitorer eller aktivatorer for en given molekylær mål eller cellulære funktion, er en meget følsom og pålidelig analyse nødvendig for at skelne mellem bona fide hits og falske positiver. Så hvad gør en god analyse? Kvaliteten af ​​en given assay skal først bedømt af signal-til-støj-forhold (reflekteret gennem en Z faktor). 8 andet bør målrettet virkning eller målet af skærmen være klart fastlagt. For eksempel kan funktionelle cellebaserede indebære betydelige fordele for receptor screening i modsætning til et assay specifikt designet til at vurdere ligand-receptorbinding. Grunden til dette er, at sidstnævnte tilgang ikke kan skelne mellem agonist- og antagonist-ligander.ss = &quot;xref&quot;&gt; 9 I modsætning hertil en celle tilgang vil sandsynligvis være mere effektive som receptor funktion kan direkte vurderes i et biologisk fænotype (proliferation, cellecyklusstop, apoptose, og / eller differentiering). Dog skal det bemærkes, at biokemiske analyser kan give betydelige fordele i forhold fænotypiske analyser, som de krænke en bestemt intracellulær mål. Et godt optimeret biokemisk assay vil generelt have mindre data scatter end et fænotypisk screening mens bagefter forenkle opklaring af den molekylære mekanisme lægemiddel virkningsmekanisme. Men den største ulempe ved målrettede eller biokemiske assays er chancen for at amplificere antallet af falske positive tilfælde, der kan påvirke ikke-specifikke mål, når det afprøves i et biologisk system (tab af specificiteten oprindelig gennemført den biokemiske assay). 10 Selv om en veletableret afskæringspunkt mellem negative og positive hits kan minimere antallet af falske positive in den primære screening, anvendelsen af ​​et fysiologisk relevant system, efterligne den native cellulære miljø såsom intakte celler, hele væv eller hele dyret fortsat kernen i assaydesignet pendul. Derfor fænotypisk screening giver bly opdagelse med ønskelige biologiske / fænotypiske virkninger for sygdomme uden identificerede lægemiddelkandidater uden at have forudgående kendskab til forbindelsens aktivitet eller virkemåde. 11 Den heri Undersøgelsen vedrører udvikling og afprøvning af en optimeret og reproducerbar fænotypisk screening baseret på to vigtige komponenter: en kommercielt tilgængelig musemodel og et grupperet sub-familie af kemiske forbindelser. Med hensyn til dyremodel, assayet bygger på anvendelsen af lymfocytter afledt fra en musestamme (Nur77 GFP) huser et bakterielt kunstigt kromosom, som indeholder en kassette, i hvilken ekspression af det grønt fluorescerende protein (GFP) drives af the Nur77 promotoren. 12 Kendetegnet for denne stimulering er baseret på det faktum, at Nur77 er et umiddelbart tidligt gen opreguleres efter T-celle-receptor (TCR) eller B-celle-receptor (BCR) stimulation. 12 Med hensyn til den screeningsmetode selv, blev en fremgangsmåde, der anvendes til at bidrage til at undgå screening af trivielle analoger samtidig minimere den nødvendige tid til at vurdere et stort kemisk bibliotek (&gt; 10 5 forbindelser). For at gøre dette, blev en database over kemiske forbindelser udvalgt af medicinske kemikere anvender virtuelle screening-værktøjer udnyttes til at identificere topologisk lignende forbindelser ved hjælp af kendte aktive frø strukturer som henvisninger. Denne fremgangsmåde tillod os at screene 4,398 forbindelser, der repræsenterer en samlet bibliotek med over 136.000 kemiske enheder.

<table border="0" cellpadding="0" cellspacing="0" width="725">
	<tbody>
		<tr height="64">
			<td height="64" style="height:64px;width:211px;">Nur77GFP mice</td>
			<td style="width:157px;">The Jackson Laboratory</td>
			<td style="width:135px;">Mouse strain No. 016617</td>
			<td style="width:224px;">An in house colony was established at our animal facility&#160;</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">96 wells-U culture plates, sterile</td>
			<td style="width:157px;">VWR International</td>
			<td style="width:135px;">10062-902</td>
			<td style="width:224px;">T-cell activation using the magnetic beads</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:211px;">70&#181;m cell strainer, sterile</td>
			<td style="width:157px;">Corning Inc.</td>
			<td style="width:135px;">352350</td>
			<td style="width:224px;">Generation of splenocytes cell suspension</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:211px;">5 ml polystyrene round bottom tubes, sterile</td>
			<td style="width:157px;">Corning Inc.</td>
			<td style="width:135px;">352058</td>
			<td style="width:224px;">Generation of splenocytes cell suspension</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:211px;">50 ml polypropylene conical bottom tubes, sterile</td>
			<td style="width:157px;">VWR International</td>
			<td style="width:135px;">89039-656&#160;</td>
			<td style="width:224px;">Generation of splenocytes cell suspension</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:211px;">10 ml syringe without needle, sterile</td>
			<td style="width:157px;">Becton, Dickinson and Company</td>
			<td style="width:135px;">305482</td>
			<td style="width:224px;">To mash the spleen</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;">T-25 culture flask</td>
			<td style="width:157px;">Greiner Bio-One</td>
			<td style="width:135px;">690 175</td>
			<td style="width:224px;">To incudabte B cells during activation</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:211px;">5 ml cell culture dish, sterile</td>
			<td style="width:157px;">Greiner Bio-One</td>
			<td style="width:135px;">627 160</td>
			<td style="width:224px;">To mash the spleen</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:211px;">Penicillin- Streptomycin (10,000 U/mL)</td>
			<td style="width:157px;">WISENT Inc.</td>
			<td style="width:135px;">450-200-EL&#160;&#160;</td>
			<td style="width:224px;">Component of the splenocyte media</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:211px;">RPMI 1600 with sodium bicarbonate and L- glutamine</td>
			<td style="width:157px;">WISENT Inc.</td>
			<td style="width:135px;">350-002-CL&#160;</td>
			<td style="width:224px;">Component of the splenocyte media</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:211px;">MEM non-essential amino acids</td>
			<td style="width:157px;">WISENT Inc.</td>
			<td style="width:135px;">321-010-EL&#160;</td>
			<td style="width:224px;">Component of the splenocyte media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">HEPES free acid 1 M</td>
			<td style="width:157px;">WISENT Inc.</td>
			<td style="width:135px;">330-050-EL&#160;</td>
			<td style="width:224px;">Component of the splenocyte media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Sodium pyruvate solution (100mM)</td>
			<td style="width:157px;">WISENT Inc.</td>
			<td style="width:135px;">600-110-EL&#160;</td>
			<td style="width:224px;">Component of the splenocyte media</td>
		</tr>
		<tr height="72">
			<td height="72" style="height:72px;width:211px;">Fetal Bovine Serum (FBS)&#160;</td>
			<td style="width:157px;">WISENT Inc.</td>
			<td style="width:135px;">080-910&#160;</td>
			<td style="width:224px;">Component of the splenocyte media and flow-cytometry buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Phosphate buffered saline (PBS)</td>
			<td style="width:157px;">WISENT Inc.</td>
			<td style="width:135px;">311-010-CL</td>
			<td style="width:224px;">Component of flow-cytometry buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">2-Mercaptoethanol (55mM)</td>
			<td style="width:157px;">Thermo Fisher Scientific</td>
			<td style="width:135px;">21985-023</td>
			<td style="width:224px;">Component of the splenocyte media</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:211px;">T- Cells isolation kit</td>
			<td style="width:157px;">Stemcell Technologies</td>
			<td style="width:135px;">19851</td>
			<td style="width:224px;">To isolate T cells</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:211px;">B- Cells isolation kit</td>
			<td style="width:157px;">Stemcell Technologies</td>
			<td style="width:135px;">19854</td>
			<td style="width:224px;">To isolate B cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Mouse T-Activator CD3/CD28 superparamagnetic beads</td>
			<td style="width:157px;">Thermo Fisher Scientific</td>
			<td style="width:135px;">11452D</td>
			<td style="width:224px;">To activate T cells&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Cell isolation magnet</td>
			<td style="width:157px;">Stemcell Technologies</td>
			<td style="width:135px;">18000</td>
			<td style="width:224px;">To isolate T cells and remove the magnetic beads&#160;</td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:211px;">AffiniPure F(ab&#39;)2 Fragment Goat Anti-Mouse IgG + IgM (H+L)</td>
			<td style="width:157px;">Jackson ImmunoResearch Laboratories, Inc.</td>
			<td style="width:135px;">115-006-068</td>
			<td style="width:224px;">To stimulate B cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Recombinant Murine IL-7</td>
			<td style="width:157px;">Peprotech</td>
			<td style="width:135px;">217-17&#160;</td>
			<td style="width:224px;">To support T-cell survival during activation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Recombinant CD40L</td>
			<td style="width:157px;">R&#38;D Systems</td>
			<td style="width:135px;">8230-CL/CF</td>
			<td style="width:224px;">To stimulate B cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Anti-mouse CD3 antibody</td>
			<td style="width:157px;">BD Pharmingen</td>
			<td style="width:135px;">561799</td>
			<td style="width:224px;">To stain T cells for flow-cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">Anti-mouse CD19 antibody</td>
			<td style="width:157px;">BD Pharmingen</td>
			<td style="width:135px;">553786</td>
			<td style="width:224px;">To stain B cells for flow-cytometry</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:211px;">Biomed FXp</td>
			<td style="width:157px;">PerkinElmer Inc.</td>
			<td style="width:135px;">A31842</td>
			<td style="width:224px;">To re-suspend cells after 24 hours incubation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Opera Phenix High Content Screening System</td>
			<td style="width:157px;">PerkinElmer Inc.</td>
			<td style="width:135px;">HH14000000</td>
			<td style="width:224px;">To analyze GFP/Hoechst signal</td>
		</tr>
	</tbody>
</table>

a fluorescence-based lymphocyte assay suitable for high-throughput screening of small molecules

High-throughput screening (HTS) er for tiden grundpillen for identifikation af kemiske enheder stand til at modulere biokemiske reaktioner eller cellulære processer. Med fremme af bioteknologi og den høje translationelle potentiale af små molekyler, har en række innovative tilgange i lægemiddelforskning udviklet sig, hvilket forklarer den genopståede interesse i brugen af ​​HTS. Den onkologi felt er i øjeblikket den mest aktive forskningsområde til screening af narkotika, uden større gennembrud for identificering af nye immunmodulerende forbindelser målrettet mod transplantation-relaterede komplikationer eller autoimmune lidelser. Her præsenteres en roman in vitro murine fluorescerende-baserede lymfocyt assay let tilpasses til identifikation af nye immunmodulerende forbindelser. Dette assay anvender T eller B-celler afledt fra en transgen mus, hvori Nur77 promotoren driver GFP-ekspression ved T- eller B-celle-receptor-stimulering. Som GFP intensitet afspejleraktivering / transkriptionsaktivitet af målcellen, vores assay definerer et hidtil ukendt redskab til at studere virkningen af ​​given forbindelse (r) på cellulære / biologiske reaktioner. For eksempel blev en primær screening udføres ved hjælp 4,398 forbindelser i mangel af en &quot;target hypotese&quot;, som førte til identifikationen af ​​160 potentielle hits viser immunmodulerende aktiviteter. Anvendelsen af dette assay er egnet til forskningsprogrammer udforske store kemiske biblioteker inden yderligere in vitro / in vivo valideringsundersøgelser.

Design af HTS assay To vigtige faktorer blev taget i betragtning ved udformningen af ​​heri fluorescenstest. Først, vi har brug for at replikere en fysiologisk tilstand, hvor T- eller B-celle-aktivering ville repræsentere en lidelse (f.eks graft-versus-host sygdom). For det andet bør udføres vurderingen af ​​cellulær aktivering ved hjælp af en følsom og kvantitativ metode. Fluorescens er i dag en af ​​...

Watch this Scientific Journal Video about A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules at JoVE.com

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

Vi præsenterer i den aktuelle undersøgelse en hidtil ukendt fluorescens-baseret assay under anvendelse af lymfocytter afledt fra en transgen mus. Dette assay er egnet til high-throughput screening (HTS) af små molekyler forsynet med kapacitet på enten inhibere eller fremme lymfocytaktivering.

En Fluorescens-baserede lymfocyt Assay Egnet til high-throughput screening af små molekyler

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

Research

JoVE Journal

Immunology and Infection

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Kvantitativ Højkapacitetsforskning encellede cytotoksicitetsassay for T-celler

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

En mikroskopisk fænotypisk analyse til kvantificering af intracellulære mycobakterier tilpasset til screening med højt gennemløb/højt indhold

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

High-throughput Screening for Bredspektret Chemical hæmmere af RNA virus

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Højkapacitetsforskning Assay til Fænotype<em&gt; Salmonella enterica</em&gt; Typhimurium Association, Invasion, og replikering i makrofager

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

High-throughput kvantitativ real-time RT-PCR assay til bestemmelse Expression Profiler af type I og III Interferon Undertyper

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Etablering af en High-throughput Opsætning til screening små molekyler, der modulerer c-di-GMP Signalering i<em&gt; Pseudomonas aeruginosa</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Mikroskopi assays for high-throughput screening af Host faktorer involveret i<em&gt; Brucella</em&gt; Infektion af HeLa-celler

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by ...

Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein

Celle-fri biokemiske fluorometriske enzymatisk Assay for høj overførselshastighed måling af Lipid fedtstoffer i High Density Lipoprotein

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

En høj overførselshastighed Platform for Screening af Salmonella spp. /Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

A High-throughput Shigella-specific Bactericidal Assay

A High-throughput Shigella-specific Bactericidal Assay

En høj overførselshastighed Shigella-specifikke bakteriedræbende Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...