Name: Fluorescentietesten voor de studie van Mycobacterium tuberculosis Interactie met de immuunreceptor SLAMF1 (Video) | JoVE Journal
Uploaded: 2025-02-28T14:00:00.000+00:00
Duration: 7 min 42 s
Description: Leer fluorescentietesten voor de studie van Mycobacterium tuberculosis interactie met de immuunreceptor SLAMF1. Dit protocol gebruikt flowcytometrie en fluorescentiemicroscopie op humane monocyt-afgeleide macrofagen.

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</ol>

<table><tbody><tr><td>Alexa Fluor 488 secundair antilichaam</td><td>Invitrogen</td><td>A21121</td><td>Voor fluorescentiemicroscopie</td></tr><tr><td>anti-SLAMF1 FITC antilichaam&nbsp;</td><td>eBioscience</td><td>11-1509-42</td><td>Voor flowcytometrie</td></tr><tr><td>anti-SLAMF1 PE antilichaam&nbsp;</td><td>BioLegend</td><td>306308</td><td>Voor flowcytometrie</td></tr><tr><td>anti-SLAMF1 primair antilichaam&nbsp;</td><td>BioLegend</td><td>306302</td><td>Voor fluorescentiemicroscopie</td></tr><tr><td>Aqua-Poly/Mount</td><td>Polysciences</td><td>18606-20</td><td>Montagemedia</td></tr><tr><td>CD14 MicroBeads</td><td>Miltenyi Biotec</td><td>130-097-052</td><td>Voor monocyten isolatie</td></tr><tr><td>Dekglasjes 12mm</td><td>HDA</td><td>-</td><td>Voor interactieonderzoek door microscopie</td></tr><tr><td>EGS</td><td>ThermoFisher Scientific&nbsp;</td><td>21565</td><td>Voor crosslinking behandeling</td></tr><tr><td>FACSCanto II</td><td>BD Biosciences</td><td>338960</td><td>Flow cytometer met BD FACSDiva software</td></tr><tr><td>Fetaal Rundserum&nbsp;</td><td>Natocor</td><td>-</td><td>Ingeactiveerd en bestraald, voor macrofagencultuur</td></tr><tr><td>Ficoll-Paque PLUS</td><td>Cytiva</td><td>17144003</td><td>Voor PBMCs scheiding</td></tr><tr><td>Fiji/ImageJ</td><td>Open Source software</td><td>-</td><td>Voor micrograafanalyse</td></tr><tr><td>FlowJo 7.6.2&nbsp;</td><td>Tree Star</td><td>-</td><td>Voor flowcytometrieanalyse</td></tr><tr><td>Formaldehyde</td><td>Merck</td><td>K47740803613</td><td>Voor crosslinking behandeling</td></tr><tr><td>Glasplaatjes</td><td>Glass Klass</td><td>-</td><td>Voor interactieonderzoek door microscopie</td></tr><tr><td>Glycine</td><td>Sigma</td><td>G8898</td><td>Voor crosslinking behandeling</td></tr><tr><td>Imager.A2</td><td>Carl Zeiss</td><td>430005-9901-000</td><td>Fluorescentiemicroscoop met Colibri 7 verlichtingsmodule</td></tr><tr><td>iMark</td><td>BIO-RAD</td><td>1681130</td><td>Microplaat absorptiemeter&nbsp;</td></tr><tr><td>L-glutamine</td><td>Sigma Aldrich</td><td>49419</td><td>Voor macrofagencultuur</td></tr><tr><td>M. tuberculosis, stam H37Rv, gamma-bestraalde hele cellen&nbsp;</td><td>BEI Resources, NIAID, NIH</td><td>NR-14819</td><td>Voor interactieonderzoek</td></tr><tr><td>M. tuberculosis, stam H37Rv, hele cel lysaat</td><td>BEI Resources, NIAID, NIH</td><td>NR-14822</td><td>Voor macrofagen stimulatie en interactieonderzoek</td></tr><tr><td>Neofuge 13R</td><td>Heal Force</td><td>Neofuge 13R</td><td>Hoge snelheid gekoelde centrifuge voor eiwitextractie</td></tr><tr><td>Penicilline/Streptomycine</td><td>Gibco</td><td>15140122</td><td>Voor macrofagencultuur</td></tr><tr><td>PMSF</td><td>ThermoFisher Scientific</td><td>36978</td><td>Voor eiwitisolatie</td></tr><tr><td>Poly-D-Lysine</td><td>Sigma Aldrich</td><td>A-003-M</td><td>Voor dekglasjesbehandeling</td></tr><tr><td>Protease Inhibitor Cocktail</td><td>Sigma Aldrich&nbsp;</td><td>P8340</td><td>Voor eiwitisolatie</td></tr><tr><td>Rhodamine B&nbsp;</td><td>Sigma Aldrich&nbsp;</td><td>21955</td><td>Voor M. tuberculosis kleuring</td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>11875093</td><td>Voor macrofagencultuur</td></tr><tr><td>Sorvall ST 16/16R centrifuge</td><td>ThermoFisher Scientific</td><td>75004240</td><td>Voor PBMCs en monocyten isolatie</td></tr></tbody></table>

fluorescentietesten voor de studie van mycobacterium tuberculosis interactie met de immuunreceptor slamf1

Fluorescence Assays for the Study of Mycobacterium tuberculosis Interaction with the Immune Receptor SLAMF1

Fluorescentietesten voor de studie van Mycobacterium tuberculosis Interactie met de immuunreceptor SLAMF1

The evaluation of direct interaction between pathogens and immune receptors usually involves sophisticated techniques or implies the use of transgenic ...

fluorescence-assays-for-study-mycobacterium-tuberculosis-interaction

Research

JoVE Journal

Immunology and Infection

Immunologie en Infectie

Source: Barbero, A. M., et.al. Fluorescence Assays for the Study of Mycobacterium tuberculosis Interaction with the Immune Receptor SLAMF1. J. Vis. Exp. ( (2025)This video demonstrates a fluorescence-based assay to study the interaction between a macrophage receptor and Mycobacterium tuberculosis antigens. Fluorescently labeled antigens on a coverslip are incubated with macrophage protein extracts, cross-linked, antibody-labeled, and visualized by overlapping fluorescence signals.

Fluorescence-Based Assay for Investigating Macrophage Receptor Interactions with Bacterial Antigens

Source: Barbero, A. M., et al. Fluorescence Assays for the Study of Mycobacterium tuberculosis Interaction with the Immune Receptor SLAMF1. J. Vis. Exp. (2025)This video demonstrates the procedure for labeling host immune receptors that bind bacterial surface antigens, enabling their detection through flow cytometry.

Analysis of Macrophage Receptor Interactions with Pathogenic Bacteria Using Flow Cytometry

This video demonstrates the reporter enzyme fluorescence (REF) imaging for in vivo detection of Mycobacterium tuberculosis infection. The infected mice are intraperitoneally injected with a substrate for REF imaging. Upon being hydrolyzed by bacterial β-lactamase enzyme, the substrate emits fluorescence, the detection of which aids evaluation of infection.

Reporter Enzyme Fluorescence Imaging for In Vivo Tracking of Bacterial Infection

We beschrijven een werkwijze voor het volgen van de endomembraan scheuren opgewekt door de intracellulaire...

Single Cell Metingen van vacuole Rupture Veroorzaakt door intracellulaire pathogenen

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

We beschrijven de optische beeldvorming van muizen besmet met Mycobacterium tuberculosis (M. tuberculosis) met behulp van verslaggever enzym fluorescentie (REF). Dit protocol vergemakkelijkt de gevoelige en specifieke detectie van M. tuberculosis in pre-klinische diermodellen voor pathogenese, therapeutics en vaccin onderzoek.

Mycobacterium tuberculosis in muizen met verslaggever enzym fluorescentie Imaging

Deze studie ontwikkelde een test voor high-throughput screening tegen Mycobacterium abscessus met een recent gecreëerde dubbele reporterstam, waarbij fluorescentie en luminescentie worden gebruikt om de levensvatbaarheid van bacteriën snel te beoordelen. Dit protocol zal relevant zijn voor onderzoekers die nieuwe geneesmiddelen willen screenen op deze resistente bacterie.

Ontwikkeling van tests voor high-throughput medicijnscreening tegen mycobacteriën

Hier presenteren we een protocol voor intravitale beeldvorming van een verzwakte, door BSL-2 goedgekeurde Mycobacterium tuberculosis. Spanning in de muislong met behulp van een permanent longvenster. Deze benadering maakt longitudinale, enkelcelresolutie-analyse mogelijk van vroege interacties tussen gastheer en pathogeen, waaronder bacillaire lokalisatie, vasculaire dynamiek en opname van macrofagen binnen dezelfde longmicroomgeving.

Intravitale beeldvorming van verzwakte auxotrofe mycobacterium tuberculosis in muislong tijdens vroege intravasculaire infectie

Leer fluorescentietesten voor de studie van Mycobacterium tuberculosis interactie met de immuunreceptor SLAMF1. Dit protocol gebruikt flowcytometrie en fluorescentiemicroscopie op humane monocyt-afgeleide macrofagen.

Fluorescentietesten voor de studie van Mycobacterium tuberculosis Interactie met de immuunreceptor SLAMF1

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