Name: visualizing the early stages of phagocytosis
Uploaded: 2017-02-03T12:30:00.000+00:00
Duration: 8 min 4 s
Description: Watch this Scientific Journal Video about Visualizing the Early Stages of Phagocytosis at JoVE.com

Ringraziamo Wendy salmone e Nicki Watson della struttura Keck al Whitehead Institute del MIT per l&#39;imaging.

1. Preparazione di DC2.4 e RAW 264.7 cellule Lines NOTA: La linea cellulare di macrofagi come RAW 264.7 e la linea di cellule dendritiche DC2.4 sono sia di origine murina, e le seguenti condizioni sono stati usati per far crescere le cellule. <ol><li> Grow RAW 264.7 cellule in DMEM (Minimal mezzo di Eagle Dulbecco) supplementato con 10% inattivato siero fetale bovino (FBS) a 37 ° C in un incubatore umidificato con 5% di CO 2. </li><li> Crescere le cellule DC2.4 in condizioni simili a quello di RAW 264.7 cellule, tranne l&#39;uso di media RPMI (Roswell Park Memorial Institute) integrata con L-glutammina, invece di DMEM. </li></ol> 2. Preparazione di coniugati fluorescenti Particolato: Beads C. albicans, Zymosan, IgG-rivestite <ol><li> Crescere Candida albicans <ol><li> Seminare C. albicans ceppo SC5314 da uno stock di glicerolo congelato in 25 ml di YPD integrato con Uri (2% Bacto-peptone, 1%estratto di lievito, 2% di glucosio e 80 ug / ml di uridina) notte a 30 ° C. NOTA: Evitare che cresce la cultura Candida più di un OD 600 del 12. </li><li> Prendere 1 ml di C. albicans e spin giù a 2.000 xg per 5 minuti a temperatura ambiente. </li><li> Aspirare il surnatante e risospendere il pellet con 1 ml di PBS. </li><li> Raccogliere il pellet per centrifugazione a 2.000 xg per 5 min a temperatura ambiente. </li><li> Ripetere il punto 2.1.4. </li><li> Calcolare la molteplicità di infezione (MOI) di C. albicans in relazione al numero dei fagociti e procedere con il test fagocitosi come descritto nella sezione 3. Una OD 600 del 1 è equivalente a 2,5 x 10 7 Candida albicans per 1 ml. NOTA: Generazione di Candida-BFP è descritto in riferimento 13. </li></ol></li><li> Preparazione di Zymosan e perline IgG-rivestite NOTA: Zymosan è un β-glucano contenentifungine preparazione carboidrati particolato derivato da Saccharomyces cerevisiae. Zymosan è noto per evocare segnali infiammatori in macrofagi e cellule dendritiche, ed è stato ampiamente utilizzato in studi fagocitosi 13, 14, 15. <ol><li> Brevemente vortice la provetta contenente il particolato, e un&#39;aliquota una quantità sufficiente per un esperimento in una provetta da 1,5 ml. NOTA: in genere usiamo rapporto 1:10 (per una cella, aggiungere 10 particelle zymosan o perline IgG-rivestite). </li><li> Aggiungere 1 ml di PBS freddo ghiaccio, e raccogliere le particelle mediante centrifugazione per 1 min a 10.000 x g. </li><li> Aspirare il surnatante e risospendere il pellet in 1 ml di PBS ghiacciato. </li><li> Ripetere i punti 2.2.2-2.2.3 NOTA: Passare al punto 2.2.5 o conservare il tubo a 4 ° C fino al momento dell&#39;uso. </li><li> Sonicare particolato per 10 sec a ultrasuoni bagno sonicatore (120 V, 50-60 kHz) per evitare unaggregazione delle particelle NY. </li></ol></li></ol> 3. fagocitosi NOTA: La fagocitosi è un processo complesso che inizia con il legame delle particelle sulla superficie cellulare dei fagociti attraverso l&#39;interazione di PRRs con ligandi sulla superficie della particella. Binding è seguito dal montaggio di actina e le sue proteine ​​associate al sito di contatto, e la formazione di una tazza fagocitaria. Il successivo smontaggio actina avviene al phagosome e provoca la engulfment completo del particolato. Di seguito si descrivono le varie fasi della fagocitosi. <ol><li> binding Assay NOTA: Lo scopo di questo esperimento è quello di monitorare il primo passo di fagocitosi che coinvolge l&#39;interazione tra i ligandi sulle particelle indicativi ei recettori sulle fagociti. <ol><li> Seme circa 5 x 10 4 cellule in una diapositiva camera in modo che siano il 60-70% confluenti al momento dell&#39;esperimento. Alterntivamente, le cellule piastra su vetrini (in 24 o formato a 12 pozzetti) o piastre a 96 pozzetti con fondo di vetro. Incubare le cellule per una notte a 37 ° C incubatore umidificato con 5% di CO 2. NOTA: Le cellule possono essere contate con emocitometro o una tecnica simile. </li><li> Posizionare il vetrino camera (o piastra) a 10 ° C per 10 min. </li><li> Rimuovere delicatamente il supporto da ciascun pozzetto con una pipetta o di aspirazione. ATTENZIONE: Eseguire questo passaggio attentamente ciascun pozzetto di un sistema coprioggetti camerata è piccolo; è facile per perturbare le cellule con la punta di una pipetta o un forte flusso d&#39;aria di un aspiratore. </li><li> Mescolare le polveri fluorescente con un mezzo di coltura cellulare (preparato come descritto sopra). Aggiungere Candida -BFP (ad una MOI di 10), fluorescenza coniugato-zymosan o lattice perline-rabbit IgG-FITC (10 particelle per cella) alle cellule. Utilizzare un volume finale di 200 microlitri mezzi per una camera bene a garantire tutte le cellule sono coperti. </li><li> Spin giù la diapositiva da camera presso 277xg per 3 min a 10 ° C. NOTA: Questo passaggio centrifugazione contribuisce ad aumentare il contatto tra le particelle e le cellule, e sincronizza il processo di associazione. </li><li> Trasferire immediatamente il vetrino camera ad un incubatore umidificato a 37 ° C con 5% di CO 2 per 5 min. </li><li> Rimuovere il vetrino da camera dal termostato e aspirare i media. </li><li> Lavare le cellule 3 volte con PBS ghiacciato. NOTA: controllare la presenza di particelle non legate con un microscopio ottico e lavare più volte con PBS, se necessario. È essenziale per rimuovere le particelle non legate dal pozzo per minimizzare il rumore di fondo durante l&#39;analisi. </li><li> Fissare cellule aggiungendo 500 microlitri 4% paraformaldeide (PFA) diluito in PBS incubando per 30 minuti a temperatura ambiente. ATTENZIONE: Questo passaggio richiede l&#39;utilizzo di PFA 4% che è altamente tossico, corrosivo, e il potenziale carcinogeno. Una corretta si deve usare cautela quando si usa questo liquido. Inoltre, proteggere i campioni dalla luce. </li><li&gt; Lavare le cellule con l&#39;aggiunta di 500 ml di PBS nella camera e aspirare lentamente la soluzione. </li><li> Ripetere 3.1.9 passo due volte. NOTA: A questo punto, riempire ogni flacone immediatamente dopo la rimozione della precedente PBS. </li><li> Arrestare il fissaggio mediante incubazione con 500 pl di 50 mM NH 4 Cl in PBS per 10 min. </li><li> Lavare le cellule due volte con PBS come descritto al punto 3.1.10. </li><li> Permeabilize cellule aggiungendo 250 microlitri tampone di legame (0,1% saponina, 0,2% BSA in PBS) per 30 min a temperatura ambiente. </li><li> Stain F-actina aggiungendo fluorescente falloidina coniugato (diluito 1: 500 in tampone vincolante). Incubare le cellule per 1 ora a temperatura ambiente. </li><li> Lavare le cellule tre volte con PBS come descritto al punto 3.1.10. </li><li> Catturare immagini utilizzando un microscopio confocale (obiettivo 63X e 402 nm e 488 nm laser) e analizzare le immagini utilizzando ImageJ 16. NOTA: legame Una particella successo è caratterizzato dalla presenza di un cu fagocitariap nel luogo dove il contatto particolato cellula fagocitica (Figura 1). Vedere il punto 3.2.5. </li></ol></li><li> Fagocitosi assorbimento Assay NOTA: Questo esperimento è stato progettato per monitorare l&#39;internalizzazione completa di particelle etichettate fluorescenza dai fagociti utilizzando la microscopia confocale (Figura 2). <ol><li> Seme circa 5 x 10 4 cellule in una diapositiva camera in modo che siano il 60-70% confluenti al momento dell&#39;esperimento. In alternativa, le cellule piastra su vetrini (in 24 o formato a 12 pozzetti) o piastre a 96 pozzetti con fondo di vetro. Incubare le cellule per una notte a 37 ° C incubatore umidificato con 5% di CO 2. NOTA: Le cellule possono essere contate con emocitometro o una tecnica simile. </li><li> Conservare le diapositive camera (o piastra) a 10 ° C per 10 min. NOTA: raffreddamento è necessario al fine di bloccare l&#39;endocitosi e fagocitosi indesiderate, e successivamente ad una fagocitosi sincronizzato. </li><li> Perfpassi ORM da 3.1.3 a 3.1.5 </li><li> Trasferire il vetrino camera ad una 37 ° C incubatore umidificato con 5% di CO 2, e incubare per diversi tempi. Fix cellule dopo l&#39;infezione 30, 60 e 90 min dopo. Osservare C. albicans iniziano a formarsi ife a livello post infezione 60 min; fagociti cominciano a mostrare la morte cellulare (pyroptosis) dopo 90 min 8. NOTA: Questa è una raccomandazione e ottimizzazione è essenziale a seconda del tipo di cellula e del particolato utilizzato. </li><li> Eseguire i passaggi da 3.1.7 a 3.1.15. Analizzare le immagini utilizzando ImageJ 16. NOTA: un assorbimento successo è caratterizzato da una interiorizzazione completa di particolato all&#39;interno del citoplasma di un fagociti (Figura 2). Colorazione fagociti con pennarello superficie cellulare come falloidina contribuisce a delineare il contorno delle cellule. È anche essenziale per eseguire z-stack per determinare se il particolato è destinata a superficie cellulare o completely interiorizzato. </li></ol></li><li> Immagini dal vivo della fagocitosi NOTA: In questo protocollo si descrive l&#39;utilizzo di microscopia confocale per catturare le diverse fasi della fagocitosi utilizzando zymosan fluorescente coniugato. Usiamo la linea di cellule dendritiche DC2.4 che esprime stabilmente mCherry-tag F-actina 8, un biosensore che mostra la distribuzione di actina filamentosa nelle cellule viventi. Poiché fagocitosi è un processo actina-driven vivo imaging in questa cella non solo permette di catturare il movimento e la dinamica di rete F-actina, ma anche di visualizzare i diversi eventi fagociti all&#39;interno delle cellule viventi (Figura 3, Film 1). <ol><li> Seme circa 5 x 10 4 cellule in una diapositiva camera ad una concentrazione in modo che siano il 60-70% confluenti al momento dell&#39;esperimento. Incubare le cellule per una notte a 37 ° C incubatore umidificato con 5% di CO 2. NOTA: Le cellule possono essere contate con emocitometro o untecnica simile. </li><li> Mantenere i vetrini camera su ghiaccio per 10 min. NOTA: raffreddamento è necessario per bloccare l&#39;assorbimento indesiderato, e di avere una fagocitosi sincronizzato. </li><li> Eseguire i passaggi da 3.1.3 a 3.1.5 </li><li> Pre-riscaldare la fase di microscopio a 37 ° C ed equilibrare la camera con 5% di CO 2. Attendere 30-45 min per la temperatura e la CO 2 si stabilizzi prima di iniziare l&#39;imaging. NOTA: Poiché i CO 2 e temperatura livelli sono critici per la fagocitosi, è importante accendere il riscaldamento del microscopio prima dell&#39;esperimento per equilibrare la camera ambientale. </li><li> Posizionare il vetrino camera contenente le cellule nel contenitore microscopio incubatore equilibrata a 37 ° C e 5% CO 2. </li><li> Esibirsi dal vivo di immagini 17, 18. NOTA: Le impostazioni per la potenza del laser (guadagno, pinhole, ecc) può variare a seconda del tipo di the microscopio e sonda fluorescente utilizzati. Pertanto, si consiglia di consultare il manuale del microscopio per la condizione ottimale per la vostra imaging dal vivo 17,18. <ol><li> Utilizzare microscopio confocale a scansione laser (e il relativo software) per l&#39;imaging dal vivo. Utilizzare obiettivo olio 63X 1.4NA con un foro stenopeico di 1-1.1 unità Airy. </li><li> Per avviare l&#39;imaging utilizzato il campo luminoso di trovare una regione rappresentante sul vetrino da camera. Avanti, selezionare il campo facendo clic sull&#39;opzione Posizione sulla software. Per rilevare GFP nella pista 1 usato il set di filtri per 488 nm laser con un divisore di fascio a 582 nm, per rilevare le lunghezze d&#39;onda tra 488 e 582 nm. </li><li> In Pista 2 selezionare un filtro set ottimale per mCherry (RFP) utilizzando un divisore di fascio a 578 nm e rilevare le lunghezze d&#39;onda tra 578 e 600 nm. Utilizzare 488 nm e 578 nm laser con il 50% di potenza laser e un&#39;uscita guadagno di 600-700. Ottenere immagini ogni 30 secondi per un totale di 90 min. </li></ol></li></ol></li></ol>

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The authors declare no conflicts of interest.

fagociti professionali, come i macrofagi e le cellule dendritiche, inghiottire e di eliminare gli agenti patogeni d&#39;invasione rendendo quindi la fagocitosi una componente importante del sistema di difesa dell&#39;ospite. Durante questo processo fagociti vengono sottoposti a riorganizzazione membrana e riarrangiamento citoscheletro alla loro superficie cellulare 8, 19, 20. Per comprendere meglio questo processo dinamico, vis...

Nonostante la continua esposizione agli agenti patogeni come batteri, virus e funghi, il nostro corpo è ben attrezzata con sistema immunitario che forniscono protezione contro le infezioni. Il sistema immunitario innato è la prima linea di difesa contro gli agenti patogeni invasori e si basa principalmente su fagociti che riconoscono e interiorizzano obiettivi stranieri. La fagocitosi è un processo cellulare evolutivamente conservato che racchiude il engulfment di particelle indesiderate superiori a 0,5 micron. Fagociti esprimono una vasta gamma di recettori del sistema immunitario (noto anche come recettori pattern recognition, PRR) sulla superficie delle cellule che consentono loro di riconoscere i modelli molecolari associati ai patogeni (PAMPs) presenti sul patogeni prima di engulfment 3. Il legame patogeni è seguito da recettore cluster sulla superficie cellulare e innesca la formazione di una tazza fagocitaria. Ciò si traduce in rimodellamento della membrana actina-driven che sporge in girol&#39;obiettivo, alla fine avvolge e pizzicando-off per formare una discreta phagosomal vacuolo 2, 5. Il phagosome matura poi e acidifica con la successiva fusione con ritardo endosomi e lisosomi che forma fagolisosoma 6. Anche se la fagocitosi è descritto come recettore-mediata e actina-event driven, questo processo si basa anche sulla modifica spazio-temporale dei lipidi che compongono la membrana plasmatica, come fosfoinositidi (PI) e sfingolipidi 7, 8. Mentre polimerizzazione actina è dettato da un accumulo locale fosfoinositolo-4,5-bisfosfato (PI (4,5) P 2) alla base della coppa fagocitica, actina depolimerizzazione dipende dalla conversione di (PI (4,5) P 2 a fosfoinositolo-3,4,5-biphosphate (PI (3,4,5) P 3) 3, 9. Entrambe le modifichesono essenziali come la prima porta a un&#39;estensione di successo del pseudopodi intorno al bersaglio e la Quest&#39;ultimo consente affondamento di particelle nel citoplasma della fagocita 10. Le cellule che hanno la capacità di fagocitare sono o fagociti professionali, come macrofagi / monociti, granulociti neutrofili /, e le cellule dendritiche (DC) o fagociti non professionisti, come fibroblasti e cellule epiteliali 11. Fagocitosi eseguita da tutti i fagociti svolge un ruolo centrale nel mantenimento dei tessuti e rimodellamento, mentre la fagocitosi eseguita da fagociti professionali è responsabile del coordinamento della risposta immunitaria innata e adattiva contro gli agenti patogeni. fagociti professionali non si limitano a fagocitare e uccidere l&#39;agente patogeno, ma anche antigeni presenti alle cellule linfoidi del sistema immunitario adattativo. Ciò contribuisce al rilascio di citochine pro-infiammatorie e ad assunzione di cellule linfoidi, dunque ail successo blocco di infezione 12. tecniche biochimiche convenzionali sono state fondamentali per acquisire conoscenze per quanto riguarda il meccanismo molecolare di diversi processi cellulari durante la fagocitosi, come modificazioni post-traslazionali e varie associazioni ad alta affinità tra proteine. Tuttavia, è difficile ottenere informazioni sulle dinamiche spaziali e temporali di eventi fagocitarie utilizzando i metodi biochimici convenzionali. l&#39;imaging cellulare dal vivo non solo ci consente di monitorare eventi cellulari in modo sensibile il tempo, ma anche ci permette di ottenere informazioni a livello di singola cellula. Qui si descrive un metodo per investigare le diverse fasi della fagocitosi, nonché di analizzare l&#39;intero processo spatiotemporally usando la microscopia confocale.

<table><tbody><tr><td>&amp;beta;-Mercaptoethanol</td><td>AppliChem</td><td>A1108</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Cell Signaling Technology&amp;nbsp;</td><td>9998</td><td></td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dilactate)</td><td>ThermoFisher</td><td>D3571</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>ThermoFisher</td><td>BP-231-1</td><td></td></tr><tr><td>DMEM (Dulbecco&amp;rsquo;s Minimal Eagle&amp;rsquo;s medium)</td><td>Gibco</td><td>11965</td><td></td></tr><tr><td>PBS, 1x (Phosphate- Buffered Saline)</td><td>Corning cellgro</td><td>21-031-CV</td><td></td></tr><tr><td>Fetal Bovine serum</td><td>Sigma Aldrich</td><td>12003C</td><td></td></tr><tr><td>FITC-coupled IgG-coated latex beads</td><td>Cayman</td><td>500290</td><td></td></tr><tr><td>L-Glutamine 200 mM (100x)</td><td>ThermoFisher</td><td>25030081</td><td></td></tr><tr><td>Paraformaldehyde Solution (4% in PBS)</td><td>Affymetrix</td><td>19943 1 LT</td><td></td></tr><tr><td>Penicillin-streptomycin (10,000 U/ml)</td><td>ThermoFisher</td><td>&lt;a href=&quot;https://www.fishersci.com/shop/products/gibco-penicillin-streptomycin-10-000-u-ml-3/15140122&quot;&gt;15-140-122&lt;/a&gt;</td><td></td></tr><tr><td>Phalloidin-Alexa Fluor 488</td><td>ThermoFisher</td><td>A12379</td><td></td></tr><tr><td>RAW 264.7 cells</td><td>ATCC</td><td>TIB-71</td><td></td></tr><tr><td>RPMI (Roswell Park Memorial Institute)</td><td>Gibco</td><td>61870</td><td></td></tr><tr><td>Saponin</td><td>Sigma Aldrich</td><td>S7900</td><td></td></tr><tr><td>Trypan blue solution (0.4% (w/v) in PBS)</td><td>Corning cellgro</td><td>MT25900CI</td><td></td></tr><tr><td>Trypsin-EDTA (1x) (0.05%)</td><td>ThermoFisher</td><td>25300054</td><td></td></tr><tr><td>Tween 20 Surfact-Amps Detergent Solution</td><td>ThermoFisher</td><td>&amp;nbsp;85114</td><td></td></tr><tr><td>Zymosan-Alexa Fluor 594</td><td>ThermoFisher</td><td>Z23374</td><td></td></tr><tr><td>Chambered 1.0 Borosilicate Coverglass system (8 chambers)</td><td>ThermoFisher</td><td>155361</td><td></td></tr><tr><td>Glasstic slide 10 with grids</td><td>Hycor</td><td>87144</td><td></td></tr></tbody></table>

visualizing the early stages of phagocytosis

Il corpo dei mammiferi è dotata di vari strati di meccanismi che contribuiscono a difendersi dalle invasioni patogeni. Fagociti professionali del sistema immunitario - come i neutrofili, cellule dendritiche e macrofagi - mantengono la capacità innata di rilevare ed eliminare tali patogeni attraverso fagocitosi 1. La fagocitosi coinvolge eventi coreografia di riorganizzazione della membrana e actina rimodellamento sulla superficie delle cellule 2, 3. I fagociti interiorizzare con successo e sradicare molecole estranee solo quando sono soddisfatte tutte le fasi della fagocitosi. Questi passaggi includono il riconoscimento e vincolante del patogeno dai recettori pattern recognition (PRR) che risiede sulla superficie cellulare, formazione di tazza di fagocitosi attraverso actina-arricchito sporgenze membranose (pseudopodi) a circondare il particolato, e scissione della phagosome seguito da phagolysosome maturazione che risultati dellauccidendo del patogeno 3, 4. Imaging e quantificazione delle varie fasi della fagocitosi è strumentale per chiarire i meccanismi molecolari di questo processo cellulare. La presente manoscritto riporta metodi per studiare le diverse fasi della fagocitosi. Descriviamo un approccio microscopio-based per visualizzare e quantificare la formazione coppa fagocitica vincolante, e l&#39;interiorizzazione di particolato dai fagociti. Come si verifica quando la fagocitosi recettori innate su fagociti incontrano ligandi su una particella bersaglio più grande di 0,5 micron, le analisi che presentiamo qui comprendono l&#39;uso di patogeni funghi Candida albicans e altre particelle, come zymosan e perline IgG-rivestite.

Metodo Microscopio a base di monitorare le diverse fasi della fagocitosi è presentato. Vengono visualizzati i diversi eventi durante la fagocitosi delle varie particelle fluorescenti da parte delle cellule DC2.4. Utilizzando le tecniche qui descritte, abbiamo studiato il ruolo degli sfingolipidi nelle prime fasi della fagocitosi. A questo scopo, DC2.4 cellule dendritiche geneticamente carenti di Sptlc2, l&#39;enzima che catalizza la prima e limitante passo nella via di biosintesi degli sfingolipidi, sono stati utilizza...

Watch this Scientific Journal Video about Visualizing the Early Stages of Phagocytosis at JoVE.com

Visualizing the Early Stages of Phagocytosis

Here we describe a microscope-based technique to visualize and quantify the early cascades of events during phagocytosis of pathogens such as the fungi Candida albicans and particulates that are larger than 0.5 &#181;m including zymosan and IgG-coated beads.

Visualizzare le fasi iniziali della fagocitosi

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune ...

visualizing-the-early-stages-of-phagocytosis

Research

JoVE Journal

Biology

16.5K Views.  Oregon Health & Science University. Here we describe a microscope-based technique to visualize and quantify the early cascades of events during phagocytosis of pathogens such as the fungi Candida albicans and particulates that are larger than 0.5 µm including zymosan and IgG-coated beads.

Video: Visualizing the Early Stages of Phagocytosis

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

Live-cellula Microscopia Video di funghi patogeni fagocitosi

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Ricerca

Immunologia e infezioni

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

Studio di fagolisosoma Biogenesis nei macrofagi in diretta

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

Alta Tecnica Throughput fluorometrica per la valutazione dei macrofagi fagocitosi e polimerizzazione

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or neutralize the ingested material, or to present it as antigens and initiate adaptive immune responses. The pH of phagosomes is a critical parameter regulating fission or fusion with endomembranes and activation of proteolytic enzymes, events that allow the phagocytic vacuole to mature into a degradative organelle. In addition, translocation of H+ is required for the production of high levels of reactive oxygen species (ROS), which are essential for efficient killing and signaling to other host tissues. Many intracellular pathogens subvert phagocytic killing by limiting phagosomal acidification, highlighting the importance of pH in phagosome biology. Here we describe a ratiometric method for measuring phagosomal pH in neutrophils using fluorescein isothiocyanate (FITC)-labeled zymosan as phagocytic targets, and live-cell imaging. The assay is based on the fluorescence properties of FITC, which is quenched by acidic pH when excited at 490 nm but not when excited at 440 nm, allowing quantification of a pH-dependent ratio, rather than absolute fluorescence, of a single dye. A detailed protocol for performing in situ dye calibration and conversion of ratio to real pH values is also provided. Single-dye ratiometric methods are generally considered superior to single wavelength or dual-dye pseudo-ratiometric protocols, as they are less sensitive to perturbations such as bleaching, focus changes, laser variations, and uneven labeling, which distort the measured signal. This method can be easily modified to measure pH in other phagocytic cell types, and zymosan can be replaced by any other amine-containing particle, from inert beads to living microorganisms. Finally, this method can be adapted to make use of other fluorescent probes sensitive to different pH ranges or other phagosomal activities, making it a generalized protocol for the functional imaging of phagosomes.

Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy.

Misurare phagosome pH Raziometrico microscopia a fluorescenza

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or ...

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force for plasma membrane extension around the particle. In addition, efficient engulfment of large material relies on focal exocytosis of intracellular compartments. This process is highly dynamic and numerous molecular players have been described to have a role during phagocytic cup formation. The precise regulation in time and space of all of these molecules, however, remains elusive. In addition, the last step of phagosome closure has been very difficult to observe because inhibition by RNA interference or dominant negative mutants often results in stalled phagocytic cup formation. 
We have set up a dedicated experimental approach using total internal reflection fluorescence microscopy (TIRFM) combined with epifluorescence to monitor step by step the extension of pseudopods and their tips in a phagosome growing around a particle loosely bound to a coverslip. This method allows us to observe, with high resolution the very tips of the pseudopods and their fusion during closure of the phagosome in living cells for two different fluorescently tagged proteins at the same time.

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM

We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

&quot;Phagosome chiusura Assay&quot; per visualizzare phagosome Formazione in tre dimensioni utilizzando la riflessione totale interna fluorescente Microscopy (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements ...

Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular compartment where the killing and digestion of engulfed particles take place, is the main arena for neutrophil pathogen killing that requires tight regulation. Phagosomal pH is one aspect that is carefully controlled, in turn regulating antimicrobial protease activity. Many fluorescent pH-sensitive dyes have been used to visualize the phagosomal environment. S-1 has several advantages over other pH-sensitive dyes, including its dual emission spectra, its resistance to photo-bleaching, and its high pKa. Using this method, we have demonstrated that the neutrophil phagosome is unusually alkaline in comparison to other phagocytes. By using different biochemical conjugations of the dye, the phagosome can be delineated from the cytoplasm so that changes in the size and shape of the phagosome can be assessed. This allows for further monitoring of ionic movement.

This manuscript describes a simple method to measure the phagosomal pH and area as well as the cytoplasmic pH of human and mouse neutrophils using the ratiometric indicator seminaphthorhodafluor (SNARF)-1, or S-1. This is achieved using live-cell confocal fluorescence microscopy and image analysis.

Imaging dei neutrofili phagosome e nel citoplasma utilizzo di un indicatore di pH Raziometrico

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

Time-lapse Imaging 3D della fagocitosi dai macrofagi del topo

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up Leishmania parasites by phagocytosis, consisting of recognizing ligands on parasite surfaces by multiple host cell receptors. Binding of Leishmania to macrophage membranes occurs through complement receptor type 1 (CR1) and complement receptor type 3 (CR3) and Pattern Recognition Receptors. Lipophosphoglycan (LPG) and 63 kDa glycoprotein (gp63) are the main ligands involved in macrophage-Leishmania interactions. Following the initial recognition of parasite ligands by host cell receptors, parasites become internalized, survive, and multiply within parasitophorous vacuoles. The maturation process of Leishmania-induced vacuoles involves the acquisition of molecules from intracellular vesicles, including monomeric G protein Rab 5 and Rab 7, lysosomal associated membrane protein 1 (LAMP-1), lysosomal associated membrane protein 2 (LAMP-2), and microtubule-associated protein 1A/1B-light chain 3 (LC3).
Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells using confocal microscopy, including (i) binding (ii) internalization, and (iii) phagosome maturation. By adding to the body of knowledge surrounding these determinants of infection outcome, we hope to improve the understanding of the pathogenesis of Leishmania infection and support the eventual search for novel chemotherapeutic targets.

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

The mechanism associated with phagocytosis in Leishmania infection remains poorly understood. Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells.

Studiare la fagocitosi della Leishmania usando la microscopia confocale

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up ...

Mesenchymal Stem Cell Regulation of Macrophage Phagocytosis; Quantitation and Imaging

Mesenchymal stem cells (MSC) have traditionally been studied for their regenerative properties, but more recently, their immunoregulatory characteristics have been at the forefront. They interact with and regulate immune cell activity. The focus of this study is the MSC regulation of macrophage phagocytic activity. Macrophage (M&#934;) phagocytosis is an important part of the innate immune system response to infection, and the mechanisms through which MSC modulate this response are under active investigation. Presented here is a method to study M&#934; phagocytosis of non-opsonized zymosan particles conjugated to a pH-sensitive fluorescent molecule while in co-culture with MSC. As phagocytic activity increases and the labeled zymosan particles are enclosed within the acidic environment of the phagolysosome, the fluorescence intensity of the pH-sensitive molecule increases. With the appropriate excitation and emission wavelengths, phagocytic activity is measured using a fluorescent spectrophotometer and kinetic data is presented as changes in relative fluorescent units over a 70 min period. To support this quantitative data, the change in the phagocytic activity is visualized using dynamic imaging. Results using this method demonstrate that when in co-culture, MSC enhance M&#934; phagocytosis of non-opsonized zymosan of both naive and IFN-&#947; treated M&#934;. These data add to the current knowledge of MSC regulation of the innate immune system. This method can be applied in future investigations to fully delineate the underlying cellular and molecular mechanisms.

Presented here is a protocol to quantify and produce dynamic images of mesenchymal stem cell (MSC) mediated regulation of macrophage (M&#934;) phagocytosis of non-opsonized yeast (zymosan) particles that are conjugated to a pH-sensitive fluorescent molecule.

Regolazione mesenchimale delle cellule staminali della fagocitosi dei macrofagi; Quantificazione e imaging

Mesenchymal stem cells (MSC) have traditionally been studied for their regenerative properties, but more recently, their immunoregulatory characteristics ...

Real-Time Imaging of Macrophage Phagocytosis of IgG-Opsonized Red Blood Cells

Source: Horsthemke, M., et al. <a href="https://app.jove.com/v/57566">Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages.</a> J. Vis. Exp. (2018)
This video demonstrates an assay to monitor independent macrophage-mediated red blood cell (RBC) phagocytosis events. Mouse immunoglobulin G (IgG)-opsonized human RBCs are incubated with mouse macrophages and observed under a confocal microscope. The RBCs are engulfed by macrophages via a phagocytic cup formation and targeted for lysosomal degradation.

Imaging in tempo reale della fagocitosi macrofagica dei globuli rossi opsonizzati IgG

Source: Horsthemke, M., et al. Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages. J. Vis. Exp. (2018)
This video demonstrates an assay to monitor ...