Name: Saggi di fluorescenza per lo studio dellinterazione del Mycobacterium tuberculosis con il recettore immunitario SLAMF1 (Video) | JoVE Journal
Uploaded: 2025-02-28T14:00:00.000+00:00
Duration: 7 min 42 s
Description: Scopri saggi di fluorescenza per studiare l'interazione tra Mycobacterium tuberculosis e il recettore immunitario SLAMF1 su macrofagi umani. Questo metodo aiuta a comprendere i meccanismi di riconoscimento patogeno.

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</ol>

<table><tbody><tr><td>Anticorpo secondario Alexa Fluor 488</td><td>Invitrogen</td><td>Codice: A21121</td><td>Per microscopia a fluorescenza</td></tr><tr><td>anticorpo FITC anti-SLAMF1&nbsp;</td><td>eBioscience</td><td>11-1509-42</td><td>Per citometria a flusso</td></tr><tr><td>anticorpo PE anti-SLAMF1&nbsp;</td><td>BioLeggenda</td><td>306308</td><td>Per citometria a flusso</td></tr><tr><td>anticorpo primario anti-SLAMF1&nbsp;</td><td>BioLeggenda</td><td>306302</td><td>Per microscopia a fluorescenza</td></tr><tr><td>Aqua-Poly/Montatura</td><td>Poliscienze</td><td>18606-20</td><td>Supporti di montaggio</td></tr><tr><td>Microsfere CD14</td><td>Miltenyi Biotec</td><td>130-097-052</td><td>Per l'isolamento dei monociti</td></tr><tr><td>Vetrini coprioggetti 12mm</td><td>HDA</td><td>-</td><td>Per il test di interazione mediante microscopia</td></tr><tr><td>EGS</td><td>ThermoFisher Scientific&nbsp;</td><td>21565</td><td>Per il trattamento di reticolazione</td></tr><tr><td>FACSCanto II</td><td>BD Bioscienze</td><td>338960</td><td>Citometro a flusso con software BD FACSDiva</td></tr><tr><td>Siero fetale bovino&nbsp;</td><td>Natocor</td><td>-</td><td>Inattivato e irradiato, per coltura di macrofagi</td></tr><tr><td>Ficoll-Paque PLUS</td><td>Citiva</td><td>17144003</td><td>Per la separazione delle PBMC</td></tr><tr><td>Figi/ImageJ</td><td>Software Open Source</td><td>-</td><td>Per analisi micrografiche</td></tr><tr><td>FlowJo 7.6.2&nbsp;</td><td>Stella dell'albero</td><td>-</td><td>Per analisi di citometria a flusso</td></tr><tr><td>Formaldeide</td><td>Merck</td><td>K47740803613</td><td>Per il trattamento di reticolazione</td></tr><tr><td>Vetrini</td><td>Bicchiere Klass</td><td>-</td><td>Per il test di interazione mediante microscopia</td></tr><tr><td>Glicina</td><td>Sigma</td><td>G8898</td><td>Per il trattamento di reticolazione</td></tr><tr><td>Imager.A2</td><td>Carl Zeiss</td><td>430005-9901-000</td><td>Microscopio a fluorescenza con modulo di illuminazione Colibri 7</td></tr><tr><td>iMark</td><td>BIO-RAD</td><td>1681130</td><td>Lettore di assorbanza per micropiastre&nbsp;</td></tr><tr><td>L-glutammina</td><td>Sigma Aldrich</td><td>49419</td><td>Per la coltura dei macrofagi</td></tr><tr><td>M. tuberculosis, ceppo H37Rv, cellule intere irradiate con raggi gamma&nbsp;</td><td>Risorse BEI, NIAID, NIH</td><td>NR-14819</td><td>Per saggi di interazione</td></tr><tr><td>M. tuberculosis, ceppo H37Rv, lisato a cellule intere</td><td>Risorse BEI, NIAID, NIH</td><td>NR-14822</td><td>Per la stimolazione dei macrofagi e il saggio di interazione</td></tr><tr><td>Neofuge 13R</td><td>Forza di guarigione</td><td>Neofuge 13R</td><td>Centrifuga refrigerata ad alta velocità per l'estrazione di proteine</td></tr><tr><td>Penicillina/Streptomicina</td><td>Gibco</td><td>15140122</td><td>Per la coltura dei macrofagi</td></tr><tr><td>PMSF</td><td>ThermoFisher Scientifico</td><td>36978</td><td>Per l'isolamento delle proteine</td></tr><tr><td>Poli-D-lisina</td><td>Sigma Aldrich</td><td>A-003-M</td><td>Per il trattamento dei vetrini coprioggetti</td></tr><tr><td>Cocktail di inibitori della proteasi</td><td>Sigma Aldrich&nbsp;</td><td>P8340</td><td>Per l'isolamento delle proteine</td></tr><tr><td>Rodamina B&nbsp;</td><td>Sigma Aldrich&nbsp;</td><td>21955</td><td>Per la colorazione di M. tuberculosis</td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>11875093</td><td>Per la coltura dei macrofagi</td></tr><tr><td>Centrifuga Sorvall ST 16/16R</td><td>ThermoFisher Scientifico</td><td>75004240</td><td>Per l'isolamento di PBMC e monociti</td></tr></tbody></table>

saggi di fluorescenza per lo studio dell'interazione del mycobacterium tuberculosis con il recettore immunitario slamf1

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

In questo lavoro, viene fornito un protocollo che consente la valutazione dell'interazione di M. tuberculosis con il recettore immunitario SLAMF1 nei macrofagi umani (Figura 1). A tal fine, è stato ottenuto sangue periferico da donatori sani. Quindi, le PBMC sono state separate mediante centrifugazione sul mezzo del gradiente di densità e i monociti sono stati isolati mediante selezione magnetica positiva (purezza ≥95%, Figura 2A, B). I...

Saggi di fluorescenza per lo studio dell'interazione del Mycobacterium tuberculosis con il recettore immunitario 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

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JoVE Journal

Immunology and Infection

Immunologia e Infezioni

Fonte: Barbero, A. M., et.al. Saggi di fluorescenza per lo studio dell'interazione di Mycobacterium tuberculosis con il recettore immunitario SLAMF1. J. Vis. Esp. ( (2025)Questo video mostra un saggio basato sulla fluorescenza per studiare l'interazione tra un recettore dei macrofagi e gli antigeni di Mycobacterium tuberculosis . Gli antigeni marcati fluorescentmente su un coperchio vengono incubati con estratti proteici di macrofagi, reticolati, marcati da anticorpi e visualizzati tramite...

Saggio basato sulla fluorescenza per indagare le interazioni dei recettori dei macrofagi con gli antigeni batterici

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

Questo video mostra l'imaging a fluorescenza enzimatica reporter (REF) per il rilevamento in vivo dell'infezione da Mycobacterium tuberculosis. I topi infetti vengono iniettati per via intraperitoneale con un substrato per l'imaging REF. Dopo essere stato idrolizzato dall'enzima batterico β-lattamasi, il substrato emette fluorescenza, il cui rilevamento aiuta la valutazione dell'infezione.

Imaging a fluorescenza enzimatica Reporter per il monitoraggio in vivo dell'infezione batterica

Si descrive un metodo per l'inseguimento della rottura endomembrane suscitato dai batteri...

Misure singole cellule di Rottura Vacuolar causate da patogeni intracellulari

Qui descriviamo un saggio fenotipico applicabile agli schermi ad alta produttività /alto contenuto di RNA sintetico (siRNA), composto chimico e librerie mutanti Mycobacterium tuberculosis. Questo metodo si basa sul rilevamento di Mycobacterium tuberculosis con etichetta fluorescente all'interno di una cellula ospite etichettata fluorescentmente utilizzando la microscopia confocale automatizzata.

Un test fenotipico microscopico per la quantificazione dei micobatteri intracellulari adattati per lo screening ad alta produttività / alto contenuto

Abbiamo sviluppato un sistema di screening ad alto rendimento modulare per scoprire nuovi composti contro Mycobacterium tuberculosis, il targeting intracellulare e in brodo coltura di batteri e citotossicità contro la cellula ospite di mammifero.

Sistema per l&#39;efficacia e la citotossicità Proiezione di inibitori di targeting intracellulare<em&gt; Mycobacterium tuberculosis</em

Nuovi modelli e saggi che migliorerebbero il processo di sviluppo precoce dei farmaci antitubercolari di prossima generazione sono altamente auspicabili. Qui, descriviamo un test rapido, economico e compatibile con BSL-2 per valutare l'efficacia del farmaco contro il Mycobacterium tuberculosis che può essere facilmente adattato per lo screening ad alto rendimento.

Un test compatibile high-throughput per valutare Drug efficacia contro macrofagi diversi passaggi<em&gt; Mycobacterium tuberculosis</em

Descriviamo l'imaging ottico dei topi infettati con la tubercolosi del micobatterio (M. tuberculosis) usando la fluorescenza di enzima reporter (REF). Questo protocollo facilita la rilevazione sensibile e specifica di M. tuberculosis in modelli animali preclinici per la ricerca di patogenesi, terapeutica e vaccino.

Tubercolosi del micobatterio in topi con fluorescenza enzima Reporter di imaging

Questo studio ha sviluppato un test per lo screening ad alto rendimento contro Mycobacterium abscessus con un ceppo doppio reporter di recente creazione, utilizzando la fluorescenza e la luminescenza per valutare rapidamente la vitalità batterica. Questo protocollo sarà rilevante per i ricercatori che mirano a selezionare nuovi farmaci contro questo batterio resistente ai farmaci.

Sviluppo di saggi per lo screening di farmaci ad alto rendimento contro i micobatteri

Qui presentiamo un protocollo per l'imaging intravitale di Mycobacterium tuberculosis approvato BSL-2 attenuato. Ceppo nel polmone murino utilizzando una finestra polmonare permanente. Questo approccio consente un'analisi longitudinale a risoluzione monocellulare delle prime interazioni ospite-patogeno, inclusa localizzazione bacillare, dinamica vascolare e assorbimento di macrofagi nello stesso microambiente polmonare.

Imaging intravitale dell'attenuazione dell'Auxotrophic Mycobacterium tuberculosis nel polmone murino durante l'infezione intravascolare precoce

Scopri saggi di fluorescenza per studiare l'interazione tra Mycobacterium tuberculosis e il recettore immunitario SLAMF1 su macrofagi umani. Questo metodo aiuta a comprendere i meccanismi di riconoscimento patogeno.

Saggi di fluorescenza per lo studio dell'interazione del Mycobacterium tuberculosis con il recettore immunitario SLAMF1

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