Questo studio è stato finanziato dal Canadian Institutes of Health Research (CIHR) operano Grant MOP-123419, scienze naturali e ingegneria ricerca Consiglio del Canada Discovery Grant 418194 e un ministero di Ontario di ricerca e innovazione primi Research Award a BH. DGW contribuito alcune delle immagini presentate, per l'ottimizzazione dei protocolli e la scrittura del manoscritto; Egli era finanziato da una sovvenzione di innesco della pompa dal Università di Liverpool. CY è finanziato da una borsa di studio laureato Vanier e CIHR MD/PhD Studentship. Le agenzie di finanziamento non avevano alcun ruolo nel disegno dello studio, raccolta dati e analisi, decisione di pubblicare o preparazione del manoscritto.

Gli autori non dichiarano alcun conflitto di interessi.

I metodi descritti in questo protocollo permettono la formazione immagine e la quantificazione del processo dinamico efferocytic, utilizzando approcci sia fisso-cellula e cellule vive. Questi approcci offrono diversi vantaggi rispetto ai metodi basati su citometria a flusso comunemente impiegate23,24. L'uso di dentro-fuori la macchiatura con campioni fissati fornisce una più robusta e precisa quantificazione del tasso e l'entità del fagosoma — infatti, molti ...

L'apoptosi, o morte programmata delle cellule, è un processo fisiologico altamente regolamentato che si verifica nella maggior parte degli organismi multicellulare ed è cruciale per il loro sviluppo e omeostasi1. Oltre a essere coinvolti nel turnover cellulare normale e lo sviluppo embrionale, apoptosi consente l'eliminazione delle cellule infette o danneggiate dai tessuti e possono essere attivato in risposta all'infezione, infiammazione, tumore e anche di interventi medici ad esempio, la radioterapia o steroidi1. Le cellule apoptotiche espongono "mangiare-me" segnali sulla superficie cellulare che vengono riconosciuti dai recettori su una gamma di fagociti professionali e non professionali, indicate collettivamente come "efferocytes". Impegno di questi recettori induce l'assorbimento e la degradazione della cellula apoptotica di efferocyte attraverso un processo noto come fagosoma2,3. La fosfatidilserina è il meglio caratterizzato mangiare-me segnale guida fagosoma. Normalmente è confinata all'opuscolo interno della membrana plasmatica, con apoptosi attivando un scramblase del lipido che sconvolge questa asimmetria di membrana, esponendo così la fosfatidilserina sulla superficie delle cellule4. La fosfatidilserina è trovata sulla superficie extracellulare di alcune cellule non apoptotiche, come macrofagi maturi e piastrine attivate. Tuttavia, queste cellule non sono efferocytosed a causa della presenza di segnali "non mangiarmi", come CD47, il loro cellulare superficiale5,6,7. Esposta la fosfatidilserina è riconosciuta da una matrice di efferocytic recettori espressi da efferocytes. Il legame di questi recettori di fosfatidilserina, direttamente o tramite l'ausilio di opsonine, attiva vie di segnalazione che promuovono la fagocitazione della cellula apoptotica in un vacuolo membrana-limita, chiamato il efferosome8, 9 , 10 , 11 , 12. il efferosome in sequenza si fonde con gli endosomi e lisosomi, che trasportano il macchinario molecolare necessario acidificare il efferosome e degradare l'apoptosi delle cellule di carico13,14. Una volta degradata, i materiali derivati da cellule apoptotiche sono vittime della tratta per il riciclaggio endosoma — un processo che limita la risposta immunitaria agli antigeni derivati da cellule apoptotiche, e che può consentire il recupero di sostanze nutritive dalla apoptotica delle cellule13, 15. Si verifica un errore nel fagosoma in distanza alterata delle cellule apoptotiche; Queste cellule non liquidate alla fine subiscono la necrosi secondaria. Le cellule necrotiche rilasciano contenuto citosolico pro-infiammatorie, agenti patogeni e autoantigeni nell'ambiente extracellulare, guidando così una gamma di malattie infettive, infiammatorie e autoimmuni16,17. Insieme, apoptosi e fagosoma facilita la rimozione delle cellule morte e morte e consentire il mantenimento dell'omeostasi tissutale.
Indagando i meccanismi molecolari sottostanti fagosoma richiede metodi che forniscono una chiara quantificazione dell'assorbimento delle cellule apoptotiche. Questa quantificazione è complicata dal fatto che a differenza di altri l'assorbimento meccanismi quali endocitosi e fagocitosi18,19, Fagosoma non possono provocare il engulfment cella obiettivo intatto, conseguente l'assorbimento a fasi della cellula apoptotica di efferocyte20. Il protocollo descritto nel presente documento descrive un in vitro fagosoma saggio che fornisce precisa delineazione dell'interiorizzato contro non interiorizzato porzioni delle cellule apoptotiche individuale e può essere combinato con una varietà di cellula fissa e approcci di microscopia di cellule vive. Saggi di fagocitosi tradizionale aggiungere gli anticorpi specifici per la destinazione fagocitica alla fine dell'esperimento al fine di etichettare gli obiettivi non interiorizzato, dove, come il nostro metodo differisce di etichettatura il bersaglio apoptotiche con biotina covalentemente21 , 22. mentre gli anticorpi specifici di cellule apoptotiche possono essere usati per questo test, l'approccio di biotinilazione consente per qualsiasi destinazione di proteina-cuscinetto di essere etichettato ed evita potenziali problemi con cross-reattività dell'anticorpo secondario se immunostaining viene eseguita . In particolare, delineiamo la preparazione delle cellule Jurkat apoptotiche che sono stati a doppia colorazione con un colorante di rilevamento delle cellule e la biotina. La cella traccianti consente apoptotica materiali derivati da cellule di essere monitorati durante Fagosoma, mentre biotinylation superficie consente la discriminazione di interiorizzato da porzioni non interiorizzato delle cellule apoptotiche efferocytosed. Descriviamo anche la cultura e la preparazione delle linee cellulari J774.2 e THP-1 per uso come efferocytes murine ed umane, macrofagi monocito-derivati M2 come esempio di cellula primaria fagosoma e cellule Jurkat per uso come efferocytic obiettivi. Questi metodi possono essere applicati facilmente ad altre linee cellulari o cellule primarie, a cellule bersaglio che subiscono ogni forma di morte cellulare (ad es. apoptosi, necrosi e necroptosis) che imita micron imprese che simulano le cellule apoptotiche attraverso rivestimenti del lipido o rivestimento con ligandi specifici di un recettore efferocytic di interesse.
Il metodo descritto in questo protocollo ha parecchi vantaggi sopra i metodi di base di citometria a flusso comunemente utilizzati nel campo23,24. Da imaging direttamente l'interazione delle cellule fagocita-apoptotici, combinato con etichettatura chiaro di entrambi materiale a cellule apoptotiche totale e non interiorizzato, è possono effettuare misure quantitative del fagosoma. Inoltre, l'uso di pH-insensibile fluorofori limita fattori di confondimento quali la soppressione della fluorescenza FITC e GFP a pH lisosomiale che confonde alcuni metodi alternativi25. Infine, mentre non è descritto in dettaglio, questi metodi possono essere impiegati utilizzando efferocytes esprimendo fluorescente etichettati transgeni, o con immunostaining post-fissazione, per consentire la quantificazione delle attività della molecola di segnalazione e monitoraggio della processi cellulari durante fagosoma.

<table>
	<tbody>
		<tr>
			<td>RPMI 1640 Media</td>
			<td>Wisent</td>
			<td>3500-000-EL</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM Media</td>
			<td>Wisent</td>
			<td>319-005-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Wisent</td>
			<td>080-150</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Wisent</td>
			<td>311-010-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>18 mm circular glass coverslips #1.5 thickness</td>
			<td>Electron Microscopy Sciences</td>
			<td>72290-08</td>
			<td>Size and shape of coverslip is not critical, but 18 mm fit into the wells of a standard 12-well plate which simplifies cell culture</td>
		</tr>
		<tr>
			<td>Staurosporine</td>
			<td>Cayman Chemical</td>
			<td>81590</td>
			<td>Dissolve in DMSO at 1 mM (1,000x stock solution)</td>
		</tr>
		<tr>
			<td>Annexin V-Alexa 488</td>
			<td>ThermoFisher</td>
			<td>R37174</td>
			<td></td>
		</tr>
		<tr>
			<td>EZ-Link NHS-Biotin</td>
			<td>ThermoFisher</td>
			<td>20217</td>
			<td>Store in a dessicator. Do not prepare a stock solution.</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTrace FarRed</td>
			<td>ThermoFisher</td>
			<td>C34572</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTrace Orange</td>
			<td>ThermoFisher</td>
			<td>C34851</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoescht 33342</td>
			<td>ThermoFisher</td>
			<td>62249</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-Streptavadin</td>
			<td>ThermoFisher</td>
			<td>SA1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Lympholyte-poly cell sepration medium</td>
			<td>Cedarlane Labs</td>
			<td>CL5071</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human M-CSF</td>
			<td>Peprotech</td>
			<td>200-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human IL-4</td>
			<td>Peprotech</td>
			<td>300-25</td>
			<td></td>
		</tr>
		<tr>
			<td>J774.2 Macrophage Cell Line</td>
			<td>Sigma-Aldrich</td>
			<td>85011428-1VL</td>
			<td></td>
		</tr>
		<tr>
			<td>THP-1 Human Monocyte Cell Line</td>
			<td>ATCC</td>
			<td>TIB-202</td>
			<td></td>
		</tr>
		<tr>
			<td>Jurkat T Cell Line</td>
			<td>ATCC</td>
			<td>TIB-152</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of efferocytosis by single-cell fluorescence microscopy

Studiare il regolamento del fagosoma richiede metodi che sono in grado di quantificare con precisione l'assorbimento delle cellule apoptotiche e per sondare i processi di segnalazione e cellulari che controllano fagosoma. Questa quantificazione può essere difficile da eseguire, come le cellule apoptotiche sono spesso efferocytosed frammentario, così necessitando metodi che possono delineare con precisione fra la parte di efferocytosed di un target di apoptotic contro residuo unengulfed cellulare frammenti. L'approccio descritto nel presente documento utilizza approcci dual-etichettatura per quantificare con precisione la dinamica della capacità fagosoma ed efferocytic di efferocytes quali i macrofagi. Il citosol della cellula apoptotica è marcati con un colorante di cella-tracking per abilitare il monitoraggio di tutti i materiali di derivati da cellule apoptotiche, mentre superficie biotinilazione della cellula apoptotica consente una differenziazione tra interiorizzata e non interiorizzato frazioni di cellule apoptotiche. La capacità di efferocytic di efferocytes è determinata prendendo immagini fluorescenti delle cellule dal vivo o fisse e quantificare l'importo limite contro bersagli interiorizzati, come differenziato dalla macchiatura streptavidina. Questo approccio offre diversi vantaggi rispetto a metodi quali la citometria a flusso, vale a dire la precisa delineazione di non efferocytosed contro efferocytosed apoptotica frazioni cellulari, la capacità di misurare efferocytic dinamica da microscopia di cellule vive e la capacità di eseguire studi di segnalazione cellulare in cellule che esprimono transgeni fluorescente etichettati. I metodi descritti in questo protocollo combinato, servono come base per un approccio sperimentale flessibile che può essere utilizzato per quantificare con precisione efferocytic attività e interrogare le vie di segnalazione cellulare attive durante fagosoma.

Una notte di cultura delle cellule Jurkat con risultati di staurosporine 1 µM in apoptosi di > 95% delle cellule, che possono essere confermate con Annessina V macchiatura (Figura 1). Altri tipi di cellule possono essere utilizzati per questi esperimenti, anche se la concentrazione di staurosporine e la durata del trattamento staurosporine dovranno essere ottimizzati per ogni linea cellulare. Per un rilevamento affidabile e la quantificazione del fagosoma, >...

Watch this Scientific Journal Video about Quantification of Efferocytosis by Single-cell Fluorescence Microscopy at JoVE.com

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Fagosoma, la rimozione fagocitica delle cellule apoptotiche, è necessaria per mantenere l'omeostasi ed è facilitata dalla recettori e vie di segnalazione che consentono il riconoscimento, il engulfment e interiorizzazione delle cellule apoptotiche. Qui, presentiamo un protocollo di microscopia di fluorescenza per la quantificazione del fagosoma e l'attività delle vie di segnalazione efferocytic.

Quantificazione del fagosoma da microscopia di fluorescenza di cella singola

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

quantification-of-efferocytosis-by-single-cell-fluorescence-microscopy

Research

JoVE Journal

Immunology and Infection

12.4K Views.  University of Western Ontario. Fagosoma, la rimozione fagocitica delle cellule apoptotiche, è necessaria per mantenere l'omeostasi ed è facilitata dalla recettori e vie di segnalazione che consentono il riconoscimento, il engulfment e interiorizzazione delle cellule apoptotiche. Qui, presentiamo un protocollo di microscopia di fluorescenza per la quantificazione del fagosoma e l'attività delle vie di segnalazione efferocytic.

Video: Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

Forno a microonde Assist diagnosi rapida di malattie virali piante da microscopia elettronica a trasmissione

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

Ricerca

Immunologia e infezioni

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

Singola cella Analisi Bacillus subtilis I biofilm usando la microscopia a fluorescenza e citometria a flusso

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

Visualizzazione delle risposte tossina batterica provocata con diretta microscopia a fluorescenza cellulare

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

Misure singole cellule di Rottura Vacuolar causate da patogeni intracellulari

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

Microscopia a fluorescenza metodi per determinare la vitalità di batteri in associazione con cellule di mammifero

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

Valutare l&#39;attività anti-fungina isolato Alveolar macrofagi dal Microscopia confocale

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like Shigella flexneri and Francisella novicida need to induce the rupture of the phagosome. In contrast, Salmonella typhimurium replicates in a vacuolar compartment, known as Salmonella-containing vacuole (SCV). However certain mutants of Salmonella fail to maintain SCV integrity and are thus released into the cytosol. The percentage of cytosolic vs. vacuolar bacteria on the level of single bacteria can be measured by differential permeabilization, also known as phagosome-protection assay. The approach makes use of the property of detergent digitonin to selectively bind cholesterol. Since the plasma membrane contains more cholesterol than other cellular membranes, digitonin can be used to selectively permeabilize the plasma membrane while leaving intracellular membranes intact. In brief, following infection with the pathogen expressing a fluorescent marker protein (e.g. mCherry among others), the plasma membrane of host cells is permeabilized with a short incubation in digitonin containing buffer. Cells are then washed and incubated with a primary antibody (coupled to a fluorophore of choice) directed against the bacterium of choice (e.g. anti-Salmonella-FITC) and washed again. If unmarked bacteria are used, an additional step can be done, in which all membranes are permeabilized and all bacteria stained with a corresponding antibody. Following the staining, the percentage of vacuolar and cytosolic bacteria can be quantified by FACS or microscopy by counting single or double-positive events. Here we provide experimental details for use of this technique with the bacterium Salmonella typhimurium. The advantage of this assay is that, in contrast to other assay, it provides a quantification on the level of single bacteria, and if analyzed by microscopy provides the exact number of cytosolic and vacuolar bacteria in a given cell.

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

Quantificazione dei citosolico vs. vacuolare<em&gt; Salmonella</em&gt; In primarie macrofagi di Permeabilization differenziale

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

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 ...

A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although a component of the innate immune response, NETs are implicated in a range of disease processes including autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay. Blood is collected using conventional venipuncture techniques. Neutrophils are isolated using dextran sedimentation and a density gradient using conditions optimized for dog blood. After allowing time for attachment to the wells of a 96 well plate, neutrophils are treated with NET-inducing agonists such as phorbol-12-myristate-13-acetate or platelet activating factor. DNA release is measured by the fluorescence of a cell-impermeable nucleic acid dye. This assay is a simple, inexpensive method for quantifying NET release, but NET formation rather than other causes of cell death must be confirmed with alternative methods.

Neutrophil extracellular traps (NETs) are networks of DNA, histones and neutrophil proteins. Although a component of the innate immune response, NETs are implicated in autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay.

Un semplice test di fluorescenza per la quantificazione del Canine neutrofili extracellulare Trappola di uscita

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.