This work was supported by Inserm. Marion Bouvet is a fellow of the French Ministry for Research and Technology. Annie Turkieh is a fellow granted by European Union FP7 HOMAGE (305507).

Das Protokoll folgt den Richtlinien unserer Institutionen menschlichen Forschungsethikkommission. Buffy-Coat von gesunden menschlichen Spendern wurden aus dem regionalen Blutspendezentrum (Lille, Frankreich) erhalten. Proben aus den Buffy Coats erhalten werden als Inserm Sammlung (n ° DC2010-1209) erklärt. 1. Material und Kultur Medienvorbereitung <ol><li> Verdünnen 10x Phosphatpuffer Saline (PBS) in sterilem destilliertem Wasser, um 1x PBS erhalten. </li><li> Machen RPMI 1640 Medium mit Gentamicin (40 ug / ml) und L-Glutamin (2 mM), mit und ohne 10% gepooltem Humanserum. </li></ol> 2. Primärkulturen von Monozyten abgeleitete Makrophagen (MDM) <ol><li> Verdünnte Buffycoat (25 ml) mit 1x PBS. Sorgfältig auf einem Ficoll / Leucosep Rohr und Zentrifuge laden bei 1.600 × g für 20 min bei Raumtemperatur. </li><li> Sammeln Monozyten an der Schnittstelle in ein neues Röhrchen. Waschen 3x mit 10 ml 1 × PBS mit 0,1% EthylendiamintetraEssigsäure (EDTA) durch aufeinanderfolgende Zentrifugation bei 1.000 × g, 370 xg und 160 g für 10 min jeweils, und dann einmal in 1 × PBS allein bei 160 × g für 10 min. </li><li> Zellpellet in 5 ml RPMI-1640-Medium ohne Serum und Samen, die Zellen in 35 mm-Schalen in einer Dichte von 1 x 10 6 Zellen pro Schale. </li><li> Nach der Sedimentation für 90 min im Brutschrank, den Überstand verwerfen die nicht-adhärenten Zellen enthält. Die adhärenten Zellen zu waschen, die aus Monozyten, 3 x mit je 1 ml PBS; dann 1 ml frischem Medium, enthaltend 10% (v / v) Humanserum zu den zuvor serumfreie Zellen. </li><li> Nach 6 Tagen in Kultur, um alternative differenzierten Makrophagen (M2) zu erhalten, fügen rekombinantes humanes IL-4 (15 ng / ml) und für 6 Tage beibehalten. Dann behandeln differenzierten Makrophagen mit Lipopolysaccharid (100 ng / ml) am Tag 12 für 4 h auf M1 Makrophagen zu erhalten. </li></ol> 3. Extraktion von M1 und M2 Makrophagen Proteine ​​für 2D-Elektrophorese <ol><li> Wash macrophAlter dreimal mit 25 mM Tris, pH 7,4, und Schrott in Puffer, enthaltend 30 mM Tris pH 8, 4% 3 - [(3-Cholamidopropyl) dimethylammonio] -1-propansulfonat (CHAPS), 2 M Thioharnstoff und 7 M Harnstoff. </li><li> Lyse-Zellen unter Verwendung eines Mischers für 1,5 ml Mikrozentrifugenröhrchen für 5 min in Eis und bei -20 ° C. </li><li> Bestimmen Proteinkonzentration unter Verwendung eines kommerziellen Bradford-Reagenz. Speichern 100 ul Aliquots der Proteine ​​bei -20 ° C bis zur Verwendung. </li></ol> 4. Isoelektrofokussierung HINWEIS: Führen Sie alle Markierungsverfahren in der Dunkelheit. <ol><li> Verringern 5 ug jeder Probe (M1 und M2 Makrophagen) mit 2 mM Tris (2-carboxyethyl) phosphin (TCEP) für 1 h auf 9 &amp; mgr; l mit Lysepuffer eingestellt auf 37 ° C. </li><li> Hinzufügen Cyanin 3 (Cy3) bis M1 und M2 Extrakt Sulfhydryl-reaktive Farbstoffextrakt in einer Konzentration von 0,8 nM / ug Protein. Hinzufügen Cyanin 5 (Cy5) bis M1 und M2 Extrakte in einer Konzentration von 0,8 nM / ug Protein und inkubierenfür 30 min bei 37 ° C. </li><li> Stoppen der Reaktion durch Zugabe eines gleichen Volumens von Probenpuffer, 7 M Harnstoff, 2 M Thioharnstoff, 4% CHAPS, 130 mM Dithiothreitol (DTT) und 2% pharmalytes. </li><li> Mix Cy3-markierten M1 Proben mit Cy5-markierten M2 Proben in einer Hand und Cy3-markierten M2 Proben mit Cy5-markierten M1 Proben in einer anderen Hand. </li><li> Rehydrieren einen immobilisierten pH-Gradienten (IPG) -Streifen (240 mM, pH 3-10 linearen Gradienten) mit 450 ul der markierten gemischten Proben in Puffer, enthaltend 7 M Harnstoff, 2 M Thioharnstoff, 4% CHAPS auf einer isoelektrischen Fokussierung (IEF) Zellsystem 24 h ohne irgendeine Strom. </li><li> Fokussieren bei 300 Volt (V) für 3 Stunden und dann mit einem Gradienten bis 1000 V für 6 h mit einem Gradienten bis 8000 V für 3 Stunden und schließlich bei 8000 V für 3 Stunden. </li></ol> 5. Zweite Dimension HINWEIS: Führen Sie alle Elektrophorese Verfahren in der Dunkelheit. <ol><li> Inkubieren Sie die IPG-Streifen in Äquilibrierungspuffers enthaltening 0,1 mM Tris-HCl (pH 8), 6 M Harnstoff, 2% (w / v) Natriumdodecylsulfat (SDS) und 30% (v / v) Glycerin für 10 min. </li><li> Übertragen die äquilibrierte IPG-Streifen für die zweite Dimension (SDS-Polyacrylamid-Gelelektrophorese (PAGE)) auf 12,5% PAGE-Gelen und Dichtung mit niedrigschmelzendem Agarose. </li><li> Durchführung der Elektrophorese bei 20 ° C unter Verwendung eines Ettan-Daltsix System bei einer konstanten Spannung von 70 V über Nacht mit 300 V, gefolgt, bis die Bromphenolblau-Front den Boden des Gels erreicht. </li></ol> 6. Image Acquisition und bioinformatische Analyse <ol><li> Scan Gele zwischen den beiden Niederfluoreszenzglasplatten gegossen mit einem DIGE Imager Scanner Anregungs- / Emissionswellenlängen von 532/580 nm und 633/670 nm Cy3 für Cy5, um Bilder mit einer Pixelgröße von 100 &amp; mgr; m zu ergeben. </li><li> Führen Sie die Bildanalyse mit kommerzieller Software wie vorher beschrieben 12. </li><li> Berechnen und normalisieren Spotvolumina in jedem Bild. Vergeben Sie einen normalisierten Spotvolumen als Requisitortion des Gesamtwerts jedes Spots detektiert im Gel. </li><li> Analysieren der Unterschiede im Proteinspotvolumen für jede Art von Makrophagen durch Vergleichen der normalisierten Punktvolumenwert zwischen den beiden Gruppen (M1 und M2). Betrachten Sie den Unterschied zwischen den Spotvolumina signifikant zu sein, wenn die Änderung 1,5 fache (p &lt;0,05, one-way ANOVA-Analyse). </li></ol>

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There are no declared conflict of interest.

Das hier beschriebene Protokoll Details eines Verfahrens, um die Auswirkungen verschiedener Stimuli der beiden Subtypen von Makrophagen, M1 (proinflammatorischen) und M2 (anti-inflammatorische) analysieren. Primärkulturen von M1 und M2 Makrophagen wurden aus der Differenzierung der Monozyten, erhalten wie zuvor veröffentlichten 7. Das Verfahren von 2D DIGE Gelelektrophorese erfordert spezielle Materialien und Ausrüstung, wie IEF-Zelle für Isoelektrofokussierung, low-Fluoreszen...

Makrophagen sind heterogen und Kunststoff-Zellen, die in der Lage, verschiedene funktionelle Phänotypen zu erwerben sind. In vivo werden diese Zellen auf eine Vielzahl von Mikroumwelt signalssuch mikrobielle Produkte, Cytokine, etc. 1 In vitro. Die pro-inflammatorischen Phänotyps (M1) von Makrophagen durch Lipopolysaccharid (LPS) und dem anti-inflammatorischen Phänotyps (M2) durch einige Cytokine, wie Interleukin-4 (IL-4) induziert werden. Darüber hinaus können Makrophagen aus einem aktivierten M1 bis M2 Phänotyp zu wechseln, und umgekehrt, auf spezifische Signale 2. Abhängig von der Phänotyp wird Makrophagen verschiedene Funktionen haben. M1 Makrophagen sind Zellen, die pro-inflammatorische Zytokine wie Tumornekrosefaktor α (TNF-α) zu erzeugen, um Mikroorganismen oder Tumorzellen 3 töten. Im Gegensatz dazu M2 Makrophagen verhindern, dass diese Entzündungsreaktion wie bei der Wundheilung und Fibrose durch die Herstellung anti inflammatory Faktoren wie TGF-β 3,4. Menschliche periphere mononukleäre Blutzellen von gesunden Spendern wurden durch Ficoll-Dichtegradienten-Zentrifugation, wie zuvor beschrieben 5 wird eine von Boyum 6 angepaßt isoliert. Makrophagen in Kultur kann nach 6 Tagen Primärkultur 7 in M1 oder M2-Phänotyp unterschieden werden. Analyse der Proteinexpression oder Proteinveränderungen zwischen den beiden Subtypen von Makrophagen unter verschiedenen gesteuerten Reize, wie Host Pathogenität oder mikrobielle Toxine, hilfreich sein, um die Funktionalität der pro- und anti-inflammatorischen Makrophagen zu entschlüsseln. Proteomics sind einzigartige Werkzeuge zur direkten Überwachung von Proteinen, die spezifisch nach oben oder in der menschlichen kultivierten Makrophagen unter verschiedenen Stimuli herunterreguliert sind. Fluoreszierende Farbstoffe haben einige der Einschränkungen der 2D-Gelelektrophorese, wie niedrige Empfindlichkeit und Bildanalyse 8 gelöst &lt;<html> / Sup&gt;. Die Farbstoffe reagieren mit Cystein-Reste haben die Nachweisempfindlichkeit im Vergleich zu denen der Reaktion mit Lysinresten 9 erhöht. In einer früheren Studie haben wir gezeigt, die Nützlichkeit DIGE Sättigungs Markierung für die Analyse von Proben knapp 10 gegenüber dem klassischen silbergefärbten 2D-Elektrophorese 11. Diese Technologie ist hilfreich bei der schnellen Analyse von Proteinmodifikationen zwischen den zwei Subtypen von Makrophagen oder zwischen unbehandelten und behandelten Makrophagen aus dem gleichen Subtyp. Die Vorteile dieser Technik sind Proteomik, die Zugang zu den Informationen der Proteingröße und post-translationalen Modifikationen durch Analyse der 2D-Gel-12. Es sollte berücksichtigt werden, dass dies nicht ein hoher Durchsatz-Technik entnommen werden, wodurch die Anzahl der Proben, die analysiert werden können. Die Entwicklung von Hochdurchsatz-Assays, die auf Massenspektrometrie als prüft kürzlich 13 kann dies zu verbessern. _content &quot;&gt; Hier präsentieren wir, wie man 2D DIGE Analyse aus der Proteinextraktion von kultivierten Makrophagen durch die Prozesse der Elektrophorese Isoelektrofokussierung durchzuführen, und SDS-PAGE sowie Informationen über den Nutzen einer angemessenen 2D-Software.

<table border="0" cellpadding="0" cellspacing="0" width="472">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:208px;"><a name="RANGE!A1:C43">Name</a></td>
			<td style="width:155px;">Company</td>
			<td style="width:111px;">Catalog number</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">RPMI 1640</td>
			<td>Invitrogen</td>
			<td>31870-074</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">PBX 10 X</td>
			<td>Invitrogen</td>
			<td>14200-083</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">L-glutamine-200mM-100X</td>
			<td>Invitrogen</td>
			<td>25030-024</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">gentamycin 10 mg/mL</td>
			<td>Invitrogen</td>
			<td>15710-049</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">human serum</td>
			<td>Invitrogen</td>
			<td>34005100</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ficoll d=1,077</td>
			<td>ATGC</td>
			<td>L6115</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Leucosep</td>
			<td>Dutscher</td>
			<td>16760</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">&#160;6-wells plate PRIMARIA&#160;</td>
			<td>Becton Dickinson</td>
			<td>353846</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">IL-4</td>
			<td>Promocell</td>
			<td>B-61410</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">lipopolysaccharide</td>
			<td>Sigma-Aldrich</td>
			<td>L-2654</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Giemsa</td>
			<td>Fluka</td>
			<td>48900</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">EDTA MM372,2</td>
			<td>Research Organics&#160;</td>
			<td>3.00E+01</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Filter 0,22&#181;m</td>
			<td>Millipore</td>
			<td>SCGPTORE</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">100 mL cylinder</td>
			<td>Corning</td>
			<td>430182</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TCEP</td>
			<td>Interchim</td>
			<td>UP242214</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Bradford reagent</td>
			<td>Bio-Rad</td>
			<td>5000006</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Cy3+Cy5-reactive dye</td>
			<td>GE Healthcare</td>
			<td>25-8009-83</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">IPG strip 3-10 24cm</td>
			<td>GE Healthcare</td>
			<td>17-6002-44</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Protean IEF cell</td>
			<td>Bio-Rad</td>
			<td>165-4000</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Low-melting agarose</td>
			<td>Invitrogen</td>
			<td>15517-014</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ettan-Daltsix system</td>
			<td>GE Healthcare</td>
			<td>80-6485-08</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ettan DIGE Imager scanner</td>
			<td>GE Healthcare</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Progenesis Samespot</td>
			<td>Non linear dynamics</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">50 ml tubes</td>
			<td>any supplier</td>
			<td>n/a</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">15 ml tubes</td>
			<td>any supplier</td>
			<td>n/a</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">CHAPS</td>
			<td>Sigma-Aldrich</td>
			<td>C5070</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ur&#233;e</td>
			<td>Merk</td>
			<td>108484-500</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Thiour&#233;e</td>
			<td>Sigma-Aldrich</td>
			<td>T7875</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DTT</td>
			<td>Bio-Rad</td>
			<td>1610611</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">APS</td>
			<td>Sigma-Aldrich</td>
			<td>A3678</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TEMED</td>
			<td>Sigma-Aldrich</td>
			<td>T9281</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tris Base</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tris HCl</td>
			<td>Sigma-Aldrich</td>
			<td>T3253</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Pharmalytes 3-10</td>
			<td>GE Healthcare</td>
			<td>17-0456-01</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">SDS</td>
			<td>Sigma-Aldrich</td>
			<td>L3773</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Bromophenol blue</td>
			<td>Sigma-Aldrich</td>
			<td>114391</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G6279</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Acrylamide 40%</td>
			<td>Bio-Rad</td>
			<td>161-0148</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">2D clean Up</td>
			<td>GE Healthcare</td>
			<td>80-6454-51</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G7126</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dim&#233;thylformamide</td>
			<td>Sigma-Aldrich</td>
			<td>22705-6</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">electrode wicks</td>
			<td>Bio-Rad</td>
			<td>165-4071</td>
		</tr>
	</tbody>
</table>

proteomic profiling of macrophages by 2d electrophoresis

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

Um entsprechende Differenz Proteomanalyse durchzuführen, sollte die Verarbeitung von zu analysierenden Proben überprüft werden. Im dargestellten Beispiel ist die Zellkulturqualität von Makrophagen für die morphologische und molekulare Aspekte erforderlich, wie zuvor veröffentlicht 5. Die Differenzierung von Monozyten in Makrophagen und die Homogenität der Kultur wurde durch Phasenmikroskopie verfolgt. Figur 1 zeigt ein Beispiel von primären Kulturen von M1...

Watch this Scientific Journal Video about Proteomic Profiling of Macrophages by 2D Electrophoresis at JoVE.com

Proteomic Profiling of Macrophages by 2D Electrophoresis

Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

Proteomik von Makrophagen durch 2D-Elektrophorese

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

proteomic-profiling-of-macrophages-by-2d-electrophoresis

Research

JoVE Journal

Immunology and Infection

12.5K Aufrufe.  University Lille Nord de France. Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

Serie: Proteomic Profiling of Macrophages by 2D Electrophoresis

Depletion and Reconstitution of Macrophages in Mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFN&gamma; or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).

Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.

Depletion und Rekonstitution von Makrophagen in Mäuse

Macrophages are critical players in the innate  immune response to infectious challenge or injury, initiating the innate immune  response and directing ...

Forschung

Immunologie und Infektion

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

Isolierung humaner Monozyten durch Doppel-Zentrifugation und ihre Differenzierung zu Makrophagen in Teflon-beschichteten Zellkultur-Taschen

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

Verwendung von<em&gt; Shigella flexneri</em&gt; Zum Autophagie-Zytoskelett Interaktionen zu untersuchen

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

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 ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

Hocheffiziente Transfektion von humanen THP-1 Makrophagen durch Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

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 ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

Phänotypische Charakterisierung von Makrophagen aus Rattenniere mittels Durchflusszytometrie

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

SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can characterize thousands of proteins at a time. These powerful techniques allow us to have a systemic understanding of cellular changes, especially when cells are subjected to various stimuli, such as infections, stresses, and specific test conditions. Even with recent developments, analyzing the exosomal proteome is time-consuming and often involves complex methodologies. In addition, the resultant large dataset often needs robust and streamlined analysis in order for researchers to perform further downstream studies. Here, we describe a SILAC-based protocol for characterizing the exosomal proteome when cells are infected with HIV-1. The method is based on simple isotope labeling, isolation of exosomes from differentially labeled cells, and mass spectrometry analysis. This is followed by detailed data mining and bioinformatics analysis of the proteomic hits. The resultant datasets and candidates are easy to understand and often offer a wealth of information that is useful for downstream analysis. This protocol is applicable to other subcellular compartments and a wide range of test conditions.

Here, we describe a quantitative proteomics method using the technique of stable isotope labeling by amino acids in cell culture (SILAC) to analyze the effects of HIV-1 infection on host exosomal proteomes. This protocol can be easily adapted to cells under different stress or infection conditions.

SILAC Based Proteomic Charakterisierung von Exosomen aus HIV-1-infizierten Zellen

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can ...

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.

Isolierung, Charakterisierung und Reinigung der Makrophagen aus den Geweben von Fettleibigkeit bedingte Entzündung betroffen

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

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

This protocol provides an analysis of the macrophage subpopulations in the adult mouse central nervous system by flow cytometry and is helpful for the study of multiple markers expressed by these cells.

Analyse von Mikroglia und Monozyten-abgeleiteten Makrophagen aus dem Zentralnervensystem durch Durchflusszytometrie

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...