Wir möchten den Mitgliedern des Tsirka Labor für ihre Ratschläge und hilfreiche Kommentare. Diese Arbeit wurde von R01NS42168 unterstützt auf SET, um 12PRE12060489 RB, ​​einer NSF-3MT IGERT und Turner Dissertation Stipendium für LT und NSF-3MT IGERT zu JCN.

1. Dissection (Tag 0) <ol><li> Induce Anästhesie p0-2 Maus Welpen mit Unterkühlung. Wischen Sie die Köpfe der Welpen mit einem Kimwipe in 70% Ethanol getränkt, um das Gewebe zu desinfizieren. </li><li> Entfernen Sie die Köpfe mit einer Schere, mit 4 Welpen pro 10 cm Kulturschale. Legen Sie die Köpfe in einer Petrischale mit eiskaltem Hanks Puffer. </li><li> Verankern Sie die Köpfe mit einer gebogenen Pinzette durch die Augenhöhlen und entfernen Sie vorsichtig die Haut über den Schädel mit geraden microforceps. </li><li> Entfernen Sie die Schädelknochen mit geraden microforceps, mit Vorsicht, nicht gewaltsam öffnen oder Schäden an den Rinden. Der effektivste Weg ist, um die Entfernung beginnend in der Nähe des Kleinhirns, die an der Basis des Kopfes befindet, da diese Region verworfen beginnen. </li><li> Entfernen Sie das Gehirn mit einem kleinen Spatel, und legen Sie die (4) Gehirne in einer frischen Petrischale mit eiskaltem Hanks Puffer. </li><li> Alle Schritte ab diesem Zeitpunkt nutzen microforceps und werden unter einer disse durchgeführtktion Mikroskop. Beginnend an der ventralen Seite des Gehirns, das Gewebe zu verankern, indem das Kleinhirn und sich zwei kleine Einschnitte an beiden Seiten der Mittelhirn. Achten Sie darauf, den ganzen Weg durch das Gewebe schneiden, da die Rinden der Mittelhirn in dieser Region zu decken. </li><li> Sanft necken die Mittelhirn und Kleinhirn in einem Stück aus den beiden Rinden. Die beiden sollten Kortex bilden eine konkave Form. </li><li> Trennen Sie die beiden Kortex und orientieren einem einzigen Cortex mit der medialen Seite für weitere Präparation. Der Hippocampus kann schwierig sein, zu beobachten, aber es befindet sich gegenüber dem Riechkolben, die als kleine Knoten an dem spitzen Ende des Kortex erscheint. </li><li> Entfernen Sie den Hippocampus, die einen Halbmond-Form hat. Klappen Sie den Cortex über die Rückenseite anzuzeigen. </li><li> Mit dem Riechkolben als Ausgangspunkt, entfernen Sie alle Hirnhäute und der Riechkolben sich von der Rinde. </li><li> Die seziert cortices sollte in 15 ml konische Röhrchen mit 14 ml col platziert werdend Hanks Salzlösung auf Eis. </li></ol> 2. Cell Culture (Tag 0) <ol><li> Pre-Mantel 10 cm Zellkulturschalen mit 5 pg / ml Poly-D-Lysin (PDL) in autoklaviertes Wasser für 3 Stunden verdünnt bei 37 ° C </li><li> Saugen PDL, und waschen Platten einmal mit Wasser autoklaviert. Dry Platte unter der UV in der Gewebekultur Haube für 20 min. Dieser Schritt sollte kurz vor der Zerlegung durchgeführt werden. </li><li> Aspirate Hanks Puffer aus Röhrchen mit den Rinden von 4 Gehirne jeweils gelten 4 ml 1x Trypsin / EDTA-Lösung, verreiben Gewebe mit p1000 Spitze und Ort Röhren in 37 ° C Inkubator für 15 min. </li><li> 4 ml komplette Mikroglia Medien pro Röhre, um die enzymatische Verdauung, mix, stoppen und Spin-down Inhalte bei 15K rpm für 5 min. </li><li> Saugen Sie den Überstand und wiederholen Wäsche mit 4 ml komplette Medien. Die Zellen in 10 ml komplettem Mikroglia Medien (DMEM mit 10% FBS, 1% Natriumpyruvat, 0,08% Gentamycin), und durch ein 40 micron Masche Sieb. </li><li> Platte mit einer Dichte von 8 Kortex pro 10 ml in einem 10 cm Gewebekulturplatte und Ort in einer Gewebekultur-Inkubator bei 37 ° C und 5% CO 2 (siehe Adult Mikroglia-Protokoll). </li></ol> (Tag 3) <ol><li> Ändern Medien in allen Zellkulturschalen mit kompletter Mikroglia Medien. </li></ol> (Tag 10) <ol><li> In 400 Mikroliter 60 mM Lidocain in HBSS (Mikroglia zu lösen) in Medium der 10-cm-Zellkulturplatten und Inkubation bei Raumtemperatur (RT) für 10-15 min. </li><li> Sammeln Sie die Medien / Zellsuspension von der Platte, und waschen Sie die Platte einmal mit Hanks-Puffer. Sammeln Sie die restlichen Waschpuffer Mikroglia erholen. </li><li> In 5 mM EDTA (pH 8,0) zu der Zellsuspension in einer Endkonzentration von 50 pM oder bei einer Verdünnung 1/100. </li><li> Spin down Zellsuspension bei 1.000 xg (1500 rpm) für 5 min und in 1 ml DMEM resuspendieren mit 1% FBS. </li><li> Graf Zahl der lebensfähigen Zellen (als Erwachsener pro Microglia 3.1/3.2) und Split-Zellen in Zellkulturplatten an der gewünschten experimentellen Dichte. Etwa 1x10 6 Mikroglia sind aus einer 10 cm Platte mit den Rinden von 4 Gehirne geerntet. Für Immunfluoreszenz wird eine Dichte von 2.5x10 4 Zellen pro 18 mm Deckglas empfohlen. </li><li> Lassen Sie mindestens 24 Stunden für die Mikroglia-Zellen vollständig auf ihre verzweigten, ruhenden Zustand vor zu bedienen. </li></ol> Protokoll Text: (Adult Mikroglia) 1. Gewebeentnahme <ol><li> Coat Zellkulturplatten mit Poly-D-Lysin (PDL) für 3 Stunden bei 37 ° C oder über Nacht bei 4 ° C. Waschen der Platten einmal mit autoklaviertem Wasser unmittelbar vor der Verwendung. </li><li> Euthanize Maus durch CO 2 Ersticken und festzustellen, dass die Sterbehilfe abgeschlossen ist. Reinigen des Rückenmarks mit 70% Ethanol. </li><li> Mit einer Schere schneiden Sie die Haut an der Spitze des Rückenmarks, dann fahren Sie mit dem Rückenmark ausgeschnitten aus Region T1 bis T12. Schneidendie restlichen Muskeln aus der Seiten des Rückenmarks. </li><li> Langsam senken die Wirbel mit Mikroschere wie halten Sie sanft das Rückenmark mit den Fingerspitzen. Achten Sie darauf, Punktion des Rückenmarks, während das Gewebe ist sehr weich. Langsam extrahieren das Rückenmark mit microforceps. </li><li> Tauchen Sie das Rückenmark in einer Petrischale mit eiskaltem HBSS. Mit einem Lichtmikroskop entfernen alle sichtbaren Hirnhäute mit microforceps </li><li> Schneiden Sie das Rückenmark in transversale Segmente klein genug, um mehrere Fragmente zu erzeugen. Die Dicke der Fragmente sollte nicht mehr als 2 mm, um eine effiziente Verdauung zu erleichtern. Übertragen Sie die Rückenmark Stücke zu 15 ml konischen Röhrchen mit 1 ml eiskaltem HBSS. Halten Sie das Röhrchen auf Eis, bis die Verdauung Schritt. </li></ol> 2. Verdauung von Tissue <ol><li> Saugen HBSS von 15 ml konischen Röhrchen mit Gewebe des Rückenmarks. Achten Sie darauf, jedes Stück Gewebe anzusaugen. </li><li> 1 ml Trypsin (2.5 g / L bestrahlten Schweine Trypsin und 0.2 g / L EDTA in HBSS) und bei 37 ° C inkubiert für 30 min. </li><li> So stoppen Sie die Verdauung entfernen Trypsin und 3 ml der primären Mikroglia Medium, das Serum, um die enzymatische Verdauung stoppen enthält. </li><li> Pipettieren nach oben und unten, um das Gewebe zu entfernen. Filtern Sie die Zellsuspension mit einer 40 um Zellsieb. </li></ol> 3. Zellzählung und Oberflächen <ol><li> Mix gleichen Volumen Zellsuspension und DAPI-Lösung. </li><li> Zählen von Zellen mit einer Zählkammer. </li><li> Platte mit einer Dichte zwischen 5-7x10 5 Zellen / ml in PDL beschichteten 35 x 10 mm-Schalen. Überzug mit einer niedrigeren Dichte verhindert das Zellwachstum auch wenn Zellen wurden in 12-Well-Platten, die eine kleinere Fläche haben im Vergleich zu 35 x 10 mm-Schalen kultiviert. </li></ol>

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Mikroglia modulieren CNS normale Funktionieren sowie Entzündungsreaktionen zu verschiedenen Pathologien. Functional synaptischen Umbau von Mikroglia in der Aufrechterhaltung des normalen Gehirn Homöostase 15 in Verbindung gebracht. Während der neurogenen Kaskade sie beteiligen sich an der Clearance von neuralen Vorläuferzellen aus dem Gyrus dentatus des Hippocampus 4, 16. Daher ist es notwendig, eine Kultur, bei dem Neugeborenen und Erwachsenen Mikroglia, die die große Schwade der Entwicklung ...

Mikroglia sind die Immunzellen Bewohner des ZNS, am ähnlichsten peripheren Makrophagen in Struktur und Funktion 1. Es wurde kürzlich gezeigt, dass postnatale Mikrogliazellen von primitiven myeloischen Vorläuferzellen ableiten und vor dem achten Tag der Embryogenese erzeugt, Hochkantstellen die bisherige Vorstellung, dass postnatale hämatopoetischen Vorläuferzellen die Quelle der Mikroglia im adulten Gehirn 2 sind. Sie spielen eine wichtige Rolle in verschiedenen neurologischen Erkrankungen und können schnell zu Infektionen oder Verletzungen reagieren durch Freisetzung pro-inflammatorische oder anti-inflammatorische Zytokine 3. So Mikroglia umfassen ein Standgerät im ZNS, die manipuliert, um den Krankheitsverlauf beeinflussen kann. Die Entwicklung von soliden und reproduzierbare Methoden zur Isolierung und Kultur Neugeborenen oder Erwachsenen Mikrogliazellen ist wichtig, um zukünftige Studien. Mikroglia sind bekanntermaßen kritisch Spieler in einer Reihe von Hirnerkrankungen sein. In jüngerer Zeit roles sind für die Zellen in normale Entwicklung des Gehirns und fungieren als Mikroglia Phagozytose überschüssige neuronale Vorläuferzellen aus dem Gyrus dentatus des Hippocampus 1, 4 entstehen. Mikroglia modulieren können auch mehrere neurologischen Erkrankungen, die das Rückenmark betreffen, wie z. B. MS, neuropathische Schmerzen und Verletzungen des Rückenmarks 5-7. Rückenmark Mikroglia reagieren unterschiedlich gegenüber Gehirn Mikroglia in Reaktion auf Ansteuersignale 8, 9, vermutlich aufgrund der Unterschiede in der lokalen Umgebung. Daher ist es wichtig, eine geeignete in vitro System, um Kultur und studieren Rückenmark Mikroglia. Neonatal Mikroglia produzieren deutlich mehr von der pro-inflammatorische Zytokin Stickstoffmonoxid im Vergleich zu erwachsenen Zellen nach in vitro Stimulation mit IFN-γ oder TNF-a 10,11 weiter hervorgehoben wird, dass adulte Zellen verwenden, um Mikroglia im Rahmen von bestimmten Krankheiten zu studieren. Das Protokoll beschäftigen wir im Labor auf culture Neugeborenen Mikroglia ist eine Variante des letzten Methoden Schütteln gemischt Gliakulturen in dem Bemühen, die Mikroglia der Oberfläche der Zellkulturflasche 12 entfernen zu nutzen. Wir beschreiben auch ein Verfahren zur Kultur von Mikroglia der adulten Maus Rückenmark über ein Protokoll zuerst von Yip et al 13 beschrieben ist. Diese Methode bietet einen schnelleren Weg zur Kultur adulter Zellen im Vergleich zu anderen verfügbaren Protokolle 14. Das erhaltene Präparat beträgt 70% Mikroglia, die restlichen Prozentsatz wird von Astrozyten zusammen. Obwohl die Reinheit der Kultur ist niedriger im Vergleich zu anderen veröffentlichten Verfahren 13 ist dieses Kultursystem zur Erkundung der Mikroglia in Kultur als Antwort auf verschiedene aktivierende Stimuli sowie zur Untersuchung von Krankheiten, die hauptsächlich im Bereich des Rückenmarks und nützlich, in denen ein starker Entzündungsreaktion ist ein Hauptmerkmal. Alle beschriebenen Protokolle wurden von der Stony Brook Universi genehmigtty IACUC.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		 </tr>
		 <tr>
			<td>EDTA, 5mM</td>
			<td>Invitrogen</td>
			<td>15567-028</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Lidocaine, 60mM</td>
			<td>Sigma</td>
			<td>L-5647</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trypan Blue</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trypsin/EDTA</td>
			<td>Cellgro</td>
			<td>25-052-CI</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM, 1X</td>
			<td>Cellgro</td>
			<td>10-017</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium Pyruvate</td>
			<td>Cellgro</td>
			<td>25-000-Cl</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Gentamycin Sulfate</td>
			<td>Biowittaker</td>
			<td>17-518Z</td>
			<td></td>
		 </tr>
		 <tr>
			<td>35 x 10mm tissue culture dish</td>
			<td>Falcon</td>
			<td>353001</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Poly-D-Lysine, 100 &mu;g/ml</td>
			<td></td>
			<td></td>
			<td>Dilute 1:20</td>
		 </tr>
		 <tr>
			<td>HBSS, 1X</td>
			<td>Cellgro</td>
			<td>20-023-CV</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

culturing microglia from the neonatal and adult central nervous system

Mikroglia sind die ansässigen Makrophagen-ähnlichen Zellen des zentralen Nervensystems (ZNS) und als solche haben kritisch wichtige Rolle in physiologischen und pathologischen Prozessen wie CNS Reifung in der Entwicklung, Multiple Sklerose und Rückenmarksverletzungen. Mikrogliazellen aktiviert und rekrutiert zum Handeln durch neuronale Verletzungen oder Stimulation, wie axonalen Schäden in MS oder ischämischer Hirn-Trauma von Schlaganfällen gesehen. Diese immunkompetenten Mitglieder des ZNS sind auch gedacht, um Rollen in synaptische Plastizität unter nicht-pathologischen Bedingungen haben. Wir beschäftigen Protokolle für die Kultivierung Mikroglia von den Neugeborenen und Erwachsenen Gewebe, die das Ziel, die Anzahl lebender Zellen zu maximieren bei gleichzeitiger Minimierung der Störvariablen, wie das Vorhandensein von anderen ZNS Zelltypen und Zellkultur Trümmer sind. Wir nutzen große und leicht erkennbare CNS Komponenten (zB Cortex, Rückenmark Segmente), die den gesamten Prozess durchführbar und reproduzierbar macht. Die Verwendung von adulten Zelles ist eine geeignete Alternative zur Verwendung von neonatalen Gehirn Mikroglia, da viele Erkrankungen betreffen hauptsächlich untersuchten die postnatale Rückenmark. Diese Kultur Systeme eignen sich auch zur direkten Prüfung der Wirkung von Verbindungen, die entweder hemmen oder fördern Mikroglia-Aktivierung kann. Da Mikroglia-Aktivierung kann die Ergebnisse der Krankheit in adulten ZNS gestalten, gibt es einen Bedarf für in-vitro-Systemen in der Neugeborenen und Erwachsenen Mikroglia gezüchtet und untersucht werden können.

<html> Ein Beispiel von ruhenden und aktivierten Mikrogliazellen ist in Abbildung 1 dargestellt. Die Mikroglia wurden visualisiert 24 Stunden nach der Beschichtung (Abbildung 1a) und weisen verzweigte (Ruhe) Morphologie. Die Exposition gegenüber der Grundierung Reagenz geworden bakterielles Lipopolysaccharid (LPS) zu Veränderungen in Mikroglia Morphologie der Zellen aktiviert (Abbildung 1b). Ein Beispiel für das Zählen der Zellen, für di...

Watch this Scientific Journal Video about Culturing Microglia from the Neonatal and Adult Central Nervous System at JoVE.com

Culturing Microglia from the Neonatal and Adult Central Nervous System

Wir skizzieren Methoden für die effiziente und schnelle Isolierung / Kultur lebensfähiger Mikroglia aus dem Neugeborenen Hirnrinde und erwachsenen Rückenmark. Die Präparation und Beschichtung der kortikalen Mikroglia kann innerhalb von 90 Minuten erreicht werden, mit der anschließenden Mikroglia Ernte stattfindet ~ 10 Tage nach der ersten Sektion.

Kultivierung Mikroglia aus dem Neugeborenen und Erwachsenen Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

culturing-microglia-from-the-neonatal-and-adult-central-nervous-system

Research

JoVE Journal

Immunology and Infection

27.5K Aufrufe.  Stony Brook University. Wir skizzieren Methoden für die effiziente und schnelle Isolierung / Kultur lebensfähiger Mikroglia aus dem Neugeborenen Hirnrinde und erwachsenen Rückenmark. Die Präparation und Beschichtung der kortikalen Mikroglia kann innerhalb von 90 Minuten erreicht werden, mit der anschließenden Mikroglia Ernte stattfindet ~ 10 Tage nach der ersten Sektion.

Serie: Culturing Microglia from the Neonatal and Adult Central Nervous System

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

Primäre Mikroglia Isolation vom gemischten Zellkulturen von Gliazellen Neonatal Rattenhirngewebe

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

Forschung

Immunologie und Infektion

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

Reinigung und Visualisierung von Lipopolysaccharid aus Gram-negativen Bakterien durch heiße wäßrige-Phenol-Extraktion

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-&#954;B (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-&#945; (TNF-&#945;), upon lipopolysaccharide (LPS) and Pam3CSK4 (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions.

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (Pam).

Isolation kortikaler Mikroglia mit bewahrten Immunophänotyp und Funktionalität aus Murine Neugeborene

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Entwicklung eines Systems Hepatitis-B-Virus Reporter den frühen Stadien des Replikationszyklus zu überwachen

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

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 ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

Isolieren von Lymphozyten aus der Maus kleinen intestinalen Immunsystems

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

A Positioning Device for the Placement of Mice During Intranasal siRNA Delivery to the Central Nervous System

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the central nervous system (CNS). Recent studies demonstrate the use of a peptide, RVG9R, incorporating the minimal receptor-binding domain of the Rabies virus glycoprotein, in eliciting the delivery of siRNA into neurons in the brain. In this protocol, the peptide-siRNA formulation is delivered intranasally with a pipette in the dominant hand, while the anesthetized mouse is restrained by the scruff with the nondominant hand in a "head down-and-forward position" to avoid drainage into the lung and stomach upon inhalation. This precise gripping of mice can be learned but is not easy and requires practice and skill to result in effective CNS uptake. Furthermore, the process is long-drawn, requiring about 45 min for the administration of a total volume of ~20-30 &#181;L of solution in 1-2 &#181;L droplet volumes per inhalation, with 3-4 min rest periods between each inhalation. The objective of this study is to disclose a mouse positioning device that enables the appropriate placement of mice for efficient IN administration of the peptide-siRNA formulation. Multiple features are incorporated into the design of the device, such as four or eight positioning chairs with adjustable height and tilt to restrain anesthetized mice in the head down-and-forward position, enabling easy visualization of the mice's nares and a built-in heating pad to maintain the mice's body temperatures during the procedure. Importantly, the ability to treat four or eight mice simultaneously with RVG9R-siRNA complexes in this manner enables studies on a much quicker time scale, for the testing of an IN therapeutic siRNA approach. In conclusion, this device allows for appropriate and controlled mouse head positioning for IN application of RVG9R-siRNA and other therapeutic molecules, such as nanoparticles or antibodies, for CNS delivery.

Here, we present a protocol using a mouse positioning device that enables the appropriate placement of mice for the intranasal administration of a brain-targeting peptide-siRNA formulation enabling effective gene silencing in the central nervous system.

Ein Positionierungsgerät für die Platzierung von Mäusen während der intranasalen siRNA-Lieferung an das Zentrale Nervensystem

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the ...

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

Flow Cytometric Analyse der Lymphozyteninfiltration im Zentralnervensystem während der experimentellen Autoimmunenzephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...

Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier between the periphery and the CNS. The CNS is an immunologically specialized site, and in steady state conditions, immune privilege is most evident in the CNS parenchyma. In contrast, the meninges harbor a diverse array of resident cells, including innate and adaptive immune cells. During inflammatory conditions triggered by CNS injury, autoimmunity, infection, or even neurodegeneration, peripherally derived immune cells may enter the parenchyma and take up residence within the meninges. These cells are thought to perform both beneficial and detrimental actions during CNS disease pathogenesis. Despite this knowledge, the meninges are often overlooked when analyzing the CNS compartment, because conventional CNS tissue extraction methods omit the meningeal layers. This protocol presents two distinct methods for the rapid isolation of murine CNS tissues (i.e., brain, spinal cord, and meninges) that are suitable for downstream analysis via single-cell techniques, immunohistochemistry, and in situ hybridization methods. The described methods provide a comprehensive analysis of CNS tissues, ideal for assessing the phenotype, function, and localization of cells occupying the CNS compartment under homeostatic conditions and during disease pathogenesis.

This paper presents two optimized protocols for examining resident and peripherally derived immune cells within the central nervous system, including the brain, spinal cord, and meninges. Each of these protocols helps to ascertain the function and composition of the cells occupying these compartments under steady state and inflammatory conditions.

Isolierung von Geweben des Zentralnervensystems und zugehörigen Hirnhäuten für die nachgeschaltete Analyse von Immunzellen

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier ...

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (&#62;10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells&#160;(DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.

Wirtschaftliches und effizientes Protokoll zur Isolierung und Kultivierung von aus Knochenmark gewonnenen dendritischen Zellen aus Mäusen

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method ...