Name: Ontwikkeling van tests voor high-throughput medicijnscreening tegen mycobacteriën (Video) | JoVE Journal
Uploaded: 2024-10-25T13:50:00.000+00:00
Duration: 7 min 50 s
Description: Ontdek een high-throughput screeningsprotocol voor Mycobacterium abscessus met een dubbele reporterstam en robotische automatisering. Deze methode versnelt de ontdekking van nieuwe behandelingen voor resistente infecties.

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5 minutes

<meta charset="utf8"></head><body>High-throughput screeningstest voor Mycobacterium abscessus met behulp van Dual Reporters

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</ol>

<table><tbody><tr><td>10% Neutral buffered formalin</td><td>Bio Optica</td><td>05-01005Q</td><td>fixatieoplossing</td></tr><tr><td>384-well black round bottom polypropylene not treated microplate</td><td>Corning</td><td>3658</td><td>Witte microplates kunnen worden gebruikt; polystyreen kan worden gebruikt</td></tr><tr><td>50 TS Washer</td><td>BioTek</td><td></td><td></td></tr><tr><td>Breathe-Easy sealing membrane</td><td>Sigma-Aldrich</td><td>Z380059-1PAK</td><td></td></tr><tr><td>cell::explorer</td><td>Revvity</td><td></td><td>apparatuur-afhandelingsrobot</td></tr><tr><td>Clarithromycin</td><td>Sigma-Aldrich</td><td>A3487-100MG</td><td>Opgelost in DMSO tot de gewenste concentratie</td></tr><tr><td>DMSO</td><td>Fisher Scientific</td><td>D/4121/PB15</td><td>Verdunnen in vloeibaar groeimedium tot de gewenste concentratie</td></tr><tr><td>Harmony 5.1</td><td>Revvity</td><td></td><td>analysesoftware</td></tr><tr><td>Heracell VIOS 160i CO2 Incubator, 165 L</td><td>Thermo Scientific</td><td></td><td>celincubator</td></tr><tr><td>JANUS with 4 Tip Arm and MDT Arm</td><td>Revvity</td><td></td><td>vloeistofbewaarsysteem</td></tr><tr><td>KB 8182</td><td>Termaks</td><td></td><td>incubator met roeren</td></tr><tr><td>Libra S4+</td><td>Biochrom</td><td></td><td></td></tr><tr><td>Multidrop&nbsp;Combi Reagent Dispenser</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Opera Phenix Plus</td><td>Revvity</td><td></td><td>beeldvormingssysteem</td></tr><tr><td>PhenoPlate</td><td>Revvity</td><td>6055302</td><td>Noodzakelijk voor hoge-inhoud microscoop</td></tr><tr><td>Phosphate Buffer Saline (PBS) 1x</td><td>NA</td><td>NA</td><td>wasoplossing</td></tr><tr><td>Prestwick Chemical libraries&nbsp;</td><td></td><td></td><td>Commerciële chemische bibliotheek bedoeld voor geneesmiddelhergebruik</td></tr><tr><td>Shaking incubator 3031</td><td>GFL</td><td></td><td></td></tr><tr><td>VICTOR Nivo</td><td>Revvity</td><td></td><td></td></tr><tr><td>Cell cultuur medium:</td><td></td><td></td><td>Wanneer nodig, voeg 10% van het supplement toe</td></tr><tr><td>DMEM</td><td>Gibco</td><td>10938-025</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Biowest</td><td>S1810</td><td></td></tr><tr><td>HEPES Buffer Solution (1 M)</td><td>Gibco</td><td>15630-056</td><td></td></tr><tr><td>LCCM</td><td>NA</td><td>NA</td><td>Geconditioneerd medium van L929-cellijn die Macrophage colony stimulerende factor (M-CSF) afgeeft; celcultuurmedium supplement</td></tr><tr><td>L-glutamin (200 mM)</td><td>Thermo Scientific</td><td>25030024</td><td></td></tr><tr><td>Sodium Pyruvate 100 mM (100X)</td><td>Gibco</td><td>11360-039</td><td></td></tr><tr><td>Infectinwasoplossing</td><td></td><td></td><td></td></tr><tr><td>Apirogene water</td><td></td><td></td><td>Meerdere leveranciers beschikbaar</td></tr><tr><td>Hank's Balanced Salt Solution 10x</td><td>Gibco</td><td>14060-040</td><td>Verdunnen tot 1x met apirogene water</td></tr><tr><td>Permeabiliseringsoplossing</td><td></td><td></td><td>PBS met 0,1% Triton X100</td></tr><tr><td>PBS 1x</td><td>NA</td><td>NA</td><td></td></tr><tr><td>Triton X100</td><td>Sigma-Aldrich</td><td>T8787</td><td>Andere leveranciers beschikbaar</td></tr><tr><td>Kleuroplossing</td><td></td><td></td><td>PBS met DAPI (500 ng/mL) en HCS Far Red (10 ng/mL)</td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9564</td><td></td></tr><tr><td>HCS Far Red&nbsp;</td><td>Invitrogen</td><td>H32721</td><td></td></tr><tr><td>PBS 1x</td><td>NA</td><td>NA</td><td>Meerdere leveranciers beschikbaar</td></tr><tr><td>Vloeibaar groeimedium</td><td></td><td></td><td>Wanneer nodig, voeg 10% supplement toe</td></tr><tr><td>Middlebrook 7H9 Broth</td><td>BD Difco</td><td>271310</td><td></td></tr><tr><td>Tween80</td><td>Sigma-Aldrich</td><td>P4780</td><td>0,05% eindconcentratie. Andere leveranciers beschikbaar</td></tr><tr><td>Vloeibaar groeimediumsupplement:</td><td></td><td></td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Fisher Scientific</td><td>BP9702-100</td><td></td></tr><tr><td>D-Glucose anhydrous</td><td>Fisher Scientific</td><td>G/0450/60</td><td></td></tr></tbody></table>

ontwikkeling van tests voor high-throughput medicijnscreening tegen mycobacteriën

Assay Development for High-Throughput Drug Screening Against Mycobacteria

Ontwikkeling van tests voor high-throughput medicijnscreening tegen mycobacteriën

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. ...

assay-development-for-high-throughput-drug-screening-against

Research

JoVE Journal

Immunology and Infection

Immunologie en Infectie

This video demonstrates an assay to evaluate the efficacy of drugs against Mycobacterium tuberculosis (Mtb) infection. An in vitro infection model containing Mtb/macrophage aggregates is generated that mimics in vivo infection. Upon introducing suitable antibiotics, their efficacy is tested via resuzarin assay to monitor the survival of bacteria with increasing drug concentrations.

An Assay to Evaluate Drug Efficacy against a Mycobacterium tuberculosis Infection Model

Hier beschrijven we een fenotypische test die van toepassing is op de high-throughput/high-content schermen van small-interfering synthetic RNA (siRNA), chemische verbinding en Mycobacterium tuberculosis mutant libraries. Deze methode is gebaseerd op de detectie van fluorescerend gelabelde Mycobacterium tuberculosis in fluorescerend gelabelde gastheercel met behulp van geautomatiseerde confocale microscopie.

Een microscopische fenotypische test voor de kwantificering van intracellulaire mycobacteriën aangepast voor screening met hoge doorvoer/hoge inhoud

We hebben een modulair high-throughput screening ontwikkeld voor het ontdekken van nieuwe verbindingen tegen Mycobacterium tuberculosis, intracellulaire targeting en-bouillon kweken bacteriën en cytotoxiciteit tegen de zoogdiergastheercel.

Systeem voor de werkzaamheid en cytotoxiciteit Screening van inhibitoren voor intracellulaire<em&gt; Mycobacterium tuberculosis</em

Nieuwe modellen en testen die het vroege proces van geneesmiddelontwikkeling voor de volgende generatie tuberculosebestrijdende middelen zouden verbeteren, zijn zeer wenselijk. Hier beschrijven we een snelle, goedkope en BSL-2-compatibele test om de werkzaamheid van geneesmiddelen tegen Mycobacterium tuberculosis te evalueren, die gemakkelijk kan worden aangepast voor screening met hoge doorvoer.

Een High-throughput Compatible Assay te evalueren Drug De werkzaamheid tegen macrofaag gepasseerd<em&gt; Mycobacterium tuberculosis</em

Deze methode beschrijft het gebruik van Infrared kleurstofinkt imaging systeem voor detectie van H1N1 in bronchioalveolar lavage (BAL) vloeistof van geïnfecteerde muizen op een hoog gevoeligheid. Deze methodologie kan worden uitgevoerd in een 96 - of 384-wells plaat, vereist &lt;10 pi volume van testmateriaal en heeft het potentieel voor gelijktijdige screening van meerdere pathogenen.

High-throughput Detection Methode voor Influenza Virus

We beschrijven een single-cell high-throughput assay voor cytotoxiciteit van T-cellen te meten wanneer geïncubeerd met tumor doelcellen. Deze werkwijze gebruikt een dichte, elastomeer reeks sub-nanoliter wells (~ 100.000 putjes / array) ruimtelijk beperken de T-cellen en doelcellen met gedefinieerde verhoudingen en is gekoppeld met fluorescentie microscopie effector-target conjugatie en daaropvolgende apoptose controleren.

Quantitative High-throughput Eencellige cytotoxiciteit T cellen

Drie assays, zoals de cytopathische effect (CPE) gebaseerde bepaling, dosis-respons assay en tijd-van-toevoeging (ToA) assay ontwikkeld, geoptimaliseerd, gevalideerd en toegepast op nieuwe antivirale middelen tegen Bluetongue virus (BTV) identificeren alsmede zoals het bepalen van de mogelijke mechanisme-van-actie (MoA) voor nieuw geïdentificeerde antivirale middelen.

Testsystemen voor de identificatie van nieuwe antivirale middelen tegen Bluetongue virus

In vitro assays om virusreplicatie te meten zijn sterk verbeterd door de ontwikkeling van recombinante RNA-virussen tot expressie luciferase of andere enzymen die in staat bioluminescentie. Hier hebben we detail een high-throughput screening pijplijn dat dergelijke recombinante stammen van mazelen en chikungunya virussen breed-spectrum antivirale middelen te isoleren van chemische bibliotheken combineert.

High-throughput screening voor Breed-spectrum Chemische Remmers van RNA-virussen

Hier beschrijven we cellulaire cytotoxiciteit en ronde infectiviteitsassays die zorgen voor een snelle en nauwkeurige screening van verbindingen om hun cellulaire cytotoxiciteit (CC50) bepalen en IC 50-waarden tegen WT en resistente HIV-1.

Snelle screening van HIV reverse transcriptase en integrase-remmers

Dit protocol beschrijft een qRT-PCR-assay met hoge doorvoer voor de analyse van type I en III IFN-expressiesignaturen. De assay onderscheidt verschillen van enkele basenparen tussen de zeer vergelijkbare transcripten van deze genen. Door batchassemblage en robotisch pipetteren zijn de assays consistent en reproduceerbaar.

High-throughput Kwantitatieve real-time RT-PCR assay voor het bepalen van expressieprofielen van de typen I en III interferon subtypen

Ontdek een high-throughput screeningsprotocol voor Mycobacterium abscessus met een dubbele reporterstam en robotische automatisering. Deze methode versnelt de ontdekking van nieuwe behandelingen voor resistente infecties.

Ontwikkeling van tests voor high-throughput medicijnscreening tegen mycobacteriën

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