Name: assessing anti-fungal activity of isolated alveolar macrophages by confocal microscopy
Uploaded: 2014-07-09T21:30:00
Description: Watch this Scientific Journal Video about Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy at JoVE.com

Dette arbejde blev støttet af National Institute of Allergy og infektionssygdomme Grant R01AI079253 (til BHS) og af National Cancer Institute Cancer Center Support Grant CA016056 til Roswell Park Cancer Institute.

Alle procedurer udføres på dyr i denne undersøgelse blev godkendt af Animal Care og brug Udvalg på Roswell Park Cancer Institute og overholdt alle statslige, føderale og National Institutes of Health regler. 1. In vivo makrofag Mærkning Bemærk: PHK26 er en lipofil farvestof, der vil blive taget op af fagocytiske celler både i cirkulation og i væv, når de indgives intravenøst ​​(iv). PKH26 blev anvendt til in vivo-mærkning for at mi...

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Med denne fremgangsmåde til ex vivo-analyse af den antifungale aktivitet af makrofager sammen med in vivo-svampe udfordring, vi tidligere vist, at NADPH-oxidase i makrofager spiller en vigtig rolle i forsvaret mod A. fumigatus 4.. Brugen af isolerede alveolære makrofager giver os mulighed for at fokusere på deres svampedræbende aktivitet i fravær af andre immunceller (f.eks rekrutterede neutrofiler), som forsvarer mod Aspergillus in vivo. <p class="jove_co...

Makrofager er de første-line fagocytceller der støder inhalerede mikrober. Makrofager genkende Aspergillus motiver af patogene anerkendelse receptorer indtage og begrænse væksten af inhalerede sporer (konidier), og indlede inflammatoriske reaktioner. Fagocytten NADPH-oxidase konverterer molekylært oxygen til superoxid anion og nedstrøms reaktive oxygenmellemprodukter (ROIs). Kronisk granulomatøs sygdom (CGD) er en arvelig lidelse i NADPH oxidase præget af alvorlige bakterie-og svampeinfektioner og ved overdrevne inflammatoriske reaktioner. Selv NADPH-oxidase er kritisk for den neutrofil-medierede værtsforsvar 1 - 3 betydningen af NADPH-oxidase i makrofager er ikke veldefineret. Tidligere undersøgelser har vist, at alveolære makrofager indtager og dræbe Aspergillus sporer, mens neutrofile hovedsagelig målrette hyfe fase 5.. Imidlertid har der været modstridende results med hensyn til den rolle, NADPH-oxidase i makrofag i at kontrollere væksten af A. fumigatus sporer 6, 7. Målet med denne metode var at afgrænse specifikke rolle NADPH oxidase i makrofager i formidlingen vært forsvar mod A. fumigatus sporer. Vi fandt, at NADPH-oxidase i alveolære makrofager kontrollerer væksten af fagocyterede A. fumigatus sporer 4.. For de fleste nøje modellere værtens respons på inhalerede svampe, brugte vi alveolære makrofager høstes af bronchoalveolar udskylning fra stimulerede mus umiddelbart efter aflivningen. Brugen af isolerede alveolære makrofager muligt for os at fokusere på deres svampedræbende aktivitet i fravær af andre immunceller (f.eks rekrutterede neutrofiler), der forsvarer over for Aspergillus in vivo. I denne fremgangsmåde blev alveolære makrofager farvet in vivo før høst for således at minimere mængden af post harvest manipulation. &lt;/ P&gt; En anden fordel ved denne protokol er anvendelsen af en GFP-udtrykkende A. fumigatus stamme. Denne svamp udtrykker GFP fra konidie scene gennem hyfe scenen og har ikke behov for yderligere farvning. For at opnå billeder med mere detaljeret, valgte vi konfokal mikroskopi, der gør det muligt at genopbygningen af ​​tredimensionale strukturer fra de opnåede billeder. Tredimensional rekonstruktion giver evnen til at differentiere mellem hyfer voksende omkring snarere end i eller gennem en makrofag. Konidier kan også være præcis skelnes som intracellulær versus ekstracellulære.

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			<td height="63" style="height:63px;width:216px;">PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">PKH26GL-1KT</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2,2,2 Tribromoethanol</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">T48402</td>
			<td>Used to make AvertinAnesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-methyl-2-butanol</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">A-1685</td>
			<td>Used to make AvertinAnesthetic</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Dulbecco&#39;s Phosphate Buffered Saline (without calcium and magnesium)</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
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		<tr height="25">
			<td height="25" style="height:25px;width:216px;">NH4CL</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">A0171</td>
			<td>Used to make ACK red cell lysis buffer</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">KHCO3</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">P91444</td>
			<td>Used to make ACK red cell lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">E6758</td>
			<td>Used to make ACK red cell lysis buffer</td>
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			<td height="42" style="height:42px;width:216px;">22 gauge X 1.00 inch i.v. catheter</td>
			<td style="width:71px;">BD&#160;</td>
			<td align="right" style="width:119px;">381523</td>
			<td>Insyte Autoguard Winged</td>
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			<td height="21" style="height:21px;width:216px;">insulin syringes</td>
			<td style="width:71px;"></td>
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			<td height="21" style="height:21px;width:216px;">6 ml syringes</td>
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			<td height="42" style="height:42px;width:216px;">4-way stopcock</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">50-700-077</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">suture thread</td>
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			<td height="21" style="height:21px;width:216px;">50 ml conical centrifuge tubes</td>
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			<td height="21" style="height:21px;width:216px;">gauze sponges (4 inch square)</td>
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			<td height="21" style="height:21px;width:216px;">RPMI 1640 with L-glutamine</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">10-040-CV</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:71px;">Hyclone</td>
			<td style="width:119px;">SH30396.03</td>
			<td>heat inactivated</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hema 3 Stain Set</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">22-122-911</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cytoseal 60</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;">8310-4</td>
			<td></td>
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			<td height="63" style="height:63px;width:216px;">Sabouraud Brain Heart Infusion Agar with Chloramphenicol and Gentamicin Slants</td>
			<td style="width:71px;">BD&#160;</td>
			<td align="right" style="width:119px;">297252</td>
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			<td height="63" style="height:63px;width:216px;">Aspergillus fumigatous strain expressing green fluorescent protein</td>
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			<td>provided by Dr Margo Moore, Simon Fraser University, Burnaby, BC, Canada</td>
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			<td height="42" style="height:42px;width:216px;">100 micron Cell Strainers</td>
			<td style="width:71px;">BD Falcon</td>
			<td style="width:119px;">64753-00</td>
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			<td height="21" style="height:21px;width:216px;">scissors</td>
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			<td height="21" style="height:21px;width:216px;">forceps</td>
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			<td height="42" style="height:42px;width:216px;">Biological Safety Cabinet Class II</td>
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			<td height="42" style="height:42px;width:216px;">Sorval ST40 Centrifuge with swing bucket rotor</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
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			<td height="21" style="height:21px;width:216px;">Leica DM1000 light microscope</td>
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			<td height="21" style="height:21px;width:216px;">Hemocytometer</td>
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			<td height="42" style="height:42px;width:216px;">Cytospin 2 centrifuge</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="25" style="height:25px;width:216px;">Cell Culture Incubator</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>37 &#176;C, 5%&#160; CO2</td>
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			<td height="21" style="height:21px;width:216px;">22X22 mm micro cover glass</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">48366-227</td>
			<td>glass coverslips</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">6 well Cell Culture Plates</td>
			<td style="width:71px;">Corning</td>
			<td align="right" style="width:119px;">3506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10% Neutral Buffered Formalin</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">BDH0502-1LP</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Vectashield Mounting medium with DAPI</td>
			<td style="width:71px;">Vector Laboratories</td>
			<td style="width:119px;">H-1200</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Leica TCS SP2 system with a laser point scanner</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>Mounted on DMIRE2 fluorescence microscope</td>
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assessing anti-fungal activity of isolated alveolar macrophages by confocal microscopy

Lungerne er en grænseflade, hvor værtsceller er rutinemæssigt udsættes for mikrober og mikrobielle produkter. Makrofager er de første-line fagocytceller der støder inhalerede svampe og andre mikrober. Makrofager og andre immunceller genkende Aspergillus motiver af patogene anerkendelse receptorer og indlede nedstrøms inflammatoriske reaktioner. Fagocytten NADPH oxidase genererer reaktive ilt mellemprodukter (ROIs) og er afgørende for vært forsvar. Selv NADPH-oxidase er kritisk for den neutrofil-medierede værtsforsvar 1 - 3 betydningen af NADPH-oxidase i makrofager er ikke veldefineret. Målet med denne undersøgelse var at afgrænse specifikke rolle NADPH oxidase i makrofager i formidlingen vært forsvar mod A. fumigatus. Vi fandt, at NADPH-oxidase i alveolære makrofager kontrollerer væksten af fagocyterede A. fumigatus sporer 4.. Her beskriver vi en metode til at vurdere muligheden for mus enlveolar makrofager (AMS) til at styre væksten af fagocyteret Aspergillus sporer (konidier). Alveolære makrofager farves in vivo og ti dage senere isoleres fra mus ved bronchoalveolær lavage (BAL). Makrofager udpladet på dækglas og derefter podet med grønt fluorescerende protein (GFP)-udtrykkende A. fumigatus sporer. På bestemte tidspunkter, cellerne fikseret og antallet af intakte makrofager med fagocyterede sporer vurderes af konfokal mikroskopi.

<html> Indsamling alveolær makrofag af BAL fra ustimulerede mus vil resultere i en&gt; 95% ren population baseret på cytologi. De 3 og 7 timer (figur 2) tidspunkter forud for hyfe fase af svampevækst og giver mulighed for en sammenligning af fagocytisk effektivitet konidier blandt genotyper. 14 hr tidspunkt er hvor genotyper kan sammenlignes med hensyn til evnen til at inhibere overgang fagocyteres konidier på vævet-invasiv hyfe fase (figur 3). Intakt makrofager kan identificeres ve...

Watch this Scientific Journal Video about Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy at JoVE.com

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

Vurdere antisvampeaktivitet Isoleret alveolære makrofager ved konfokal mikroskopi

En metode til at evaluere evnen hos isolerede mus alveolære makrofager til at styre væksten af fagocyterede Aspergillus sporer ved konfokal mikroskopi.

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

Research

JoVE Journal

Immunology and Infection

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 ...

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

Intravital Mikroskopi af milten: Kvantitativ analyse af Parasite mobilitet og Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Real-time Imaging af myeloide celler dynamik i<em&gt; Apc<sup&gt; Min / +</sup</em&gt; Intestinal Tumorer ved Spinning Disk Konfokal mikroskopi

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Proteomic Profiling of Macrophages by 2D Electrophoresis

Proteomisk Profilering af makrofager ved 2D elektroforese

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Generation og Identifikation af GM-CSF Afledt Alveolære-lignende makrofager og dendritiske celler fra mus Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

Fænotypisk karakterisering af makrofager fra rottenyre ved flowcytometri

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

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

Isolation, karakterisering og rensning af makrofager fra væv ramt af fedme-relaterede Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

Visualisere makrofag ekstracellulære fælder ved hjælp af konfokalmikroskopi

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolation og In Vitro kultur af Murine og menneskelige alveolær makrofager

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Time-lapse 3D Imaging af fagocytose af musen makrofager

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