Diese Studie wurde von kanadischen Institute Health Research (CIHR) Betrieb Grant MOP-123419, Naturwissenschaften und Engineering Forschung Rat von Kanada Discovery Grant 418194, Ontario Ministerium für Forschung und Innovation frühen Forschungspreis für BH finanziert. DGW trugen einige der Bilder präsentiert, um die Optimierung der Protokolle und das Schreiben des Manuskripts; Er wurde durch eine selbstansaugende Pumpe von der University of Liverpool finanziert. CY wird von einem Vanier Graduate Stipendium und CIHR MD/PhD Zugehörigkeit finanziert. Förderinstitutionen hatte keine Rolle beim Studiendesign, Datenerhebung und Analyse, Entscheidung, zu veröffentlichen oder der Manuskripterstellung.

Die Autoren erklären keinen Interessenskonflikt.

Die in diesem Protokoll beschriebenen Methoden ermöglichen die Bildgebung und Quantifizierung des dynamischen Efferocytic-Prozesses mit fest-Zelle und live-Cell Ansätzen. Diese Ansätze bieten mehrere Vorteile gegenüber den verwendeten Flow Cytometry-basierte Methoden23,24. Die Verwendung der Inside-Out-Färbung mit festen Proben bietet eine robuste und präzise Quantifizierung der Rate und das Ausmaß der Efferocytosis — in der Tat viele Flow Cytometry-basi...

Apoptose oder programmierter Zelltod ist ein stark regulierten physiologischen Prozess, der tritt in den meisten vielzelligen Organismen und ist entscheidend für ihre Entwicklung und Homöostase1. Neben der normalen Zellerneuerung und embryonale Entwicklung beteiligt, Apoptose ermöglicht die Beseitigung der infizierten oder beschädigten Zellen aus den Geweben und in Reaktion auf die Infektion, Entzündung, Krebs, und auch durch medizinische Eingriffe ausgelöst werden kann z. B. Strahlentherapie oder Steroide1. Apoptotische Zellen aussetzen "Essen-mich" Signale auf ihrer Zelloberfläche die von Rezeptoren auf eine Reihe von professionellen und nichtprofessionellen Phagozyten erkannt werden zusammenfassend als "Efferocytes" bezeichnet. Engagement dieser Rezeptoren induziert die Aufnahme und den Abbau der apoptotischen Zelle durch die Efferocyte durch einen Prozeß bekannt als Efferocytosis2,3. Phosphatidylserin ist die beste charakterisierten essen-mir signalisieren treibende Efferocytosis. Es normalerweise beschränkt sich auf die innere Broschüre der Plasmamembran, mit Apoptose ein Lipid-Scramblase das stört diese Membran-Asymmetrie, damit aussetzen Phosphatidylserin auf der Zelle Oberfläche4aktivieren. Phosphatidylserin ist auf der extrazellulären Oberfläche von einigen nicht-apoptotische Zellen wie Reifen Makrophagen gefunden und Thrombozyten aktiviert. Diese Zellen sind jedoch nicht Efferocytosed aufgrund des Vorhandenseins von "ISS mich nicht" Signale, wie z. B. CD47, auf ihrer Zelle Oberfläche5,6,7. Exponierten Phosphatidylserin wird durch eine Reihe von Efferocytic Rezeptoren geäußerten Efferocytes erkannt. Bindung dieser Rezeptoren, Phosphatidylserin, aktiviert entweder direkt oder durch die Hilfe von Opsonins, Signalwege, die die Verwicklung der apoptotischen Zelle in eine Membrane-springen Vakuole bezeichnet die Efferosome8, zu fördern 9 , 10 , 11 , 12. das Efferosome Sicherungen sequenziell mit Endosomen und Lysosomen, die liefern die molekularen Maschinen notwendig, die Efferosome Ansäuern und verschlechtern die Apoptotic Zelle Ladung13,14. Einmal abgebaut, die Apoptotic Zelle abgeleitet Materialien sind Opfer von Menschenhandel zu recycling Endosom – ein Prozess, welche Grenzen Immunantworten auf apoptotische Zelle abgeleitet Antigene, und die können für die Verwertung von Nährstoffen aus der apoptotischen Zelle13, 15. Ein Fehler im Efferocytosis führt zu eingeschränkter Clearance von apoptotischen Zellen; diese ungeklärten Zellen durchlaufen schließlich sekundären Nekrose. Nekrotische Zellen release Pro-inflammatorischen cytosolischen Inhalt, Krankheitserreger und Autoantigene in das extrazelluläre Milieu, somit eine Reichweite von infektiösen, Entzündungs-und Autoimmunkrankheiten16,17. Zusammen, Apoptose und Efferocytosis erleichtern die Entfernung der sterbende und tote Zellen und für die Aufrechterhaltung der Homöostase Gewebe erlauben.
Untersuchung der molekularen Mechanismen, die Efferocytosis erfordert Methoden, die eine klare Quantifizierung der apoptotischen Zelle Aufnahme bereitstellen. Diese Quantifizierung wird durch die Tatsache erschwert, dass im Gegensatz zu anderen Aufnahme Mechanismen wie z. B. Endozytose und Phagozytose18,19, Efferocytosis nicht die Verwicklung von intakten Zielzelle, wodurch die schrittweise Aufnahme führen kann der apoptotischen Zelle durch die Efferocyte20. Die hierin beschriebene Protokoll beschreibt einen in-vitro- Efferocytosis Assay, die genaue Abgrenzung von den verinnerlichten bietet im Vergleich zu nicht verinnerlicht Teile der einzelnen Apoptotic Zellen und kombinierbar mit einer Vielzahl von festen-Zelle und Leben-Zelle Mikroskopie Ansätze. Traditionelle Phagozytose Assays hinzufügen Antikörper spezifisch auf die phagocytic Ziel am Ende des Experiments um Ziele nicht verinnerlicht, wo, wie unsere Methode beschriften unterscheidet sich durch Kennzeichnung der Apoptotic Ziel mit kovalent verknüpft Biotin21 , 22. während apoptotischer Zellen spezifische Antikörper in diesem Assay verwendet werden können, der biotinylierungen Ansatz ermöglicht für jedes Protein-Lager Ziel gekennzeichnet werden und vermeidet mögliche Probleme mit sekundären Antikörper Kreuzreaktivität Immunostaining erfolgt . Im einzelnen erläutern wir die Vorbereitung des apoptotischen Jurkat-Zellen, die mit einer Zelle Tracking Farbstoff und Biotin Dual befleckt gewesen. Die Zelle tracking Farbstoff ermöglicht apoptotische Zelle abgeleitet Materialien, während Efferocytosis verfolgt, während Oberfläche biotinylierungen ermöglicht die Unterscheidung von aus nicht verinnerlicht Teilen des Efferocytosed Apoptotic Zellen verinnerlicht. Wir beschreiben auch die Kultur und die Vorbereitung von J774.2 und THP-1-Zell-Linien für den Einsatz als murinen und menschlichen Efferocytes, M2-Makrophagen Monocyte abgeleitet als Beispiel der Primärzelle Efferocytosis und Jurkat-Zellen für den Einsatz als Efferocytic Ziele. Diese Methoden können leicht auch auf andere Zelllinien oder Primärzellen, Zielzellen durchläuft jede Form des Zelltods (z. B. Apoptose, Nekrose und Necroptosis) und Mikron mittelständische imitiert die Apoptotic Zellen durch Lipid-Beschichtungen zu simulieren oder Beschichtung mit Liganden spezifisch für eine Efferocytic-Rezeptor von Interesse.
In diesem Protokoll beschriebenen Methode hat mehrere Vorteile gegenüber der Flow Cytometry basierend üblichen Methoden im Feld23,24. Durch bildgebende direkt die Phagozyten-apoptotische Zelle Interaktion, kombiniert mit klaren Kennzeichnung von beiden insgesamt und nicht verinnerlicht Apoptotic Zellmaterial können quantitative Maßnahmen des Efferocytosis vorgenommen werden. Darüber hinaus schränkt die Verwendung von pH-unempfindliche Fluorophore Störfaktoren wie die Unterdrückung der FITC und GFP Fluoreszenz bei lysosomalen pH, die einige alternative Methoden25verwirrt. Schließlich, während nicht im Detail beschrieben, diese Methoden eingesetzt werden mit Efferocytes mit dem Ausdruck eindringmittel beschriftet transgene oder mit Post-Fixierung Immunostaining, Quantifizierung der Signalisierung Molekül Aktivität und Überwachung ermöglicht die zelluläre Prozesse während der Efferocytosis.

<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

Studieren die Regulierung des Efferocytosis erfordert Methoden in der Lage sind, die Aufnahme von apoptotischen Zellen genau zu quantifizieren und die Signalisierung und zelluläre Prozesse zu untersuchen, die Efferocytosis zu steuern. Diese Quantifizierung kann schwierig sein, wie Apoptotic Zellen oft bruchstückhaft, Efferocytosed sind durchführen, so erfordern Methoden, die genau zwischen dem Efferocytosed Teil eines Ziels Apoptotic versus passives unengulfed Mobilfunk abgrenzen können Fragmente. Die hier beschriebenen Ansatz nutzt Dual-Kennzeichnung Ansätze, um die Dynamik des Efferocytosis und Efferocytic der Efferocytes wie z. B. Makrophagen genau zu quantifizieren. Das Zytosol der apoptotischen Zelle ist mit einem Handy-Tracking-Farbstoff zu ermöglichen eine Überwachung aller apoptotische Zelle abgeleitet Materialien, während Oberfläche biotinylierungen der apoptotischen Zelle ermöglicht eine Differenzierung zwischen verinnerlichten und nicht verinnerlicht beschriftet apoptotische Zelle Brüche. Die Efferocytic-Kapazität des Efferocytes richtet sich nach fluoreszierende Bilder live oder festen Zellen und die Menge des gebundenen versus internalisierten Ziele zu quantifizieren, wie durch Streptavidin Färbung unterschieden. Dieser Ansatz hat mehrere Vorteile gegenüber Methoden wie Durchflusszytometrie, nämlich die genaue Abgrenzung von nicht-Efferocytosed versus Efferocytosed Apoptotic Zelle Brüche, die Fähigkeit, Efferocytic Dynamik durch live-Cell-Mikroskopie, Messen und die Kapazität, Untersuchungen der zellulären Signalisierung in Zellen, die mit dem Ausdruck eindringmittel beschriftet Transgene durchzuführen. Kombiniert, dienen die in diesem Protokoll beschriebenen Methoden als Grundlage für eine flexible experimentellen Ansatz, der verwendet werden kann, um Efferocytic Tätigkeit genau zu quantifizieren und zelluläre Signalwege während der Efferocytosis aktiv zu verhören.

Über Nacht Kultur Jurkat Zellen mit 1 µM Staurosporine Ergebnisse bei der Apoptose von > 95 % der Zellen, die mit Annexin V Färbung (Abbildung 1) bestätigt werden kann. Andere Zelltypen können für diese Experimente verwendet werden, obwohl die Konzentration der Staurosporine und die Dauer der Staurosporine Behandlung für jede Zelllinie optimiert werden müssen. Für die zuverlässige Erkennung und Quantifizierung von Efferocytosis > 80 % der Zellen sol...

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

Efferocytosis, die phagocytic Entfernung der Apoptotic Zellen ist erforderlich, um die Homöostase und wird erleichtert durch die Rezeptoren und Signalwege, die für die Anerkennung, Engulfment und Internalisierung der Apoptotic Zellen ermöglichen. Hier präsentieren wir ein Fluoreszenz-Mikroskopie-Protokoll für die Quantifizierung des Efferocytosis und die Aktivität der Efferocytic Signalwege.

Quantifizierung der Efferocytosis durch einzellige Fluoreszenz-Mikroskopie

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 Aufrufe.  University of Western Ontario. Efferocytosis, die phagocytic Entfernung der Apoptotic Zellen ist erforderlich, um die Homöostase und wird erleichtert durch die Rezeptoren und Signalwege, die für die Anerkennung, Engulfment und Internalisierung der Apoptotic Zellen ermöglichen. Hier präsentieren wir ein Fluoreszenz-Mikroskopie-Protokoll für die Quantifizierung des Efferocytosis und die Aktivität der Efferocytic Signalwege.

Serie: 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.

Mikrowelle Assisted Schnelle Diagnose von Plant Virus Erkrankungen durch Transmission Electron Microscopy

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

Forschung

Immunologie und Infektion

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.

Einzel-Zell-Analyse Bacillus subtilis Biofilme mittels Fluoreszenzmikroskopie und Durchflusszytometrie

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.

Visualisierung der bakteriellen Toxin induzierten Reaktionen mit Live Cell Fluoreszenzmikroskopie

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.

Single Cell Messungen Vacuolar Rupture durch intrazelluläre Erreger verursacht

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.

Fluoreszenz-Mikroskopie-Methoden zur Bestimmung der Lebensfähigkeit der Bakterien in Verbindung mit Säugerzellen

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.

Beurteilung der Anti-Pilz-Aktivität von isolierten Alveolarmakrophagen durch konfokale Mikroskopie

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.

Die Quantifizierung der Cytosolic vs. Vacuolar<em&gt; Salmonellen</em&gt; In Primäre Makrophagen durch Differential Permeabilisierung

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.

Mess Phagosomen pH durch ratiometrische Fluoreszenzmikroskopie

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.

Eine einfache Fluoreszenztest zur Quantifizierung von Canine Neutrophil Extracellular Einklemmschutz

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.