Gefördert durch das Programm an der intramuralen Uniformed Services University.

1. Dissection of Neonatal Rattenhirngewebe <ol><li> Frost Leibovitz L-15 konditionierten Medien (Leibowitz L-15 + 0,1% BSA + 1% Pen / Strep) auf 4 ° C Warm Kulturmedium (DMEM + 10% FBS + 1% Penicillin / Streptomycin) auf 37 ° C Bereiten Sie vier Petrischalen 60x15 mm mit 4-5 ml der konditionierten L-15 Medien auf dem Eis. Sterilisieren Sie alle chirurgischen Instrumente mit 100% Ethanol. </li><li> Spülen Sie die P2 neugeborenen Welpen Rap mit 70% Ethanol. (Neonatal Rattenjungen zwischen P1-P5 Jahren können in diesem Protokoll verwendet werden.) </li><li> Schnell enthaupten ein Jungtier mit einer sterilen scharfen Schere und legen Sie den Kopf sofort in 70% Ethanol. Wiederholen Sie dies für insgesamt 5 Rattenjungen dann übertragen Köpfe in Kochsalzlösung. </li><li> Entfernen Sie die ganze Gehirne aus den Köpfen und Ort in eine Petrischale mit 4 - 5 ml L-15-Lösung (Leibowitz L-15 + 0,1% BSA + 1% Pen / Strep) auf Eis. </li><li> Wiederholen Sie die Prozeduren von 1,2 bis 1,4 für die restlichen Welpen im Wurf. </li><li> Entfernen Sie die Meningen des Gehirns und übertragen die desIRED Hirngewebe (dh Kortex, Kleinhirn ganze Gehirn, etc.) in eine neue Petrischale mit 5.4 ml L-15-Lösung (Leibowitz L-15 + 0,1% BSA + 1% Pen / Strep) auf Eis. </li></ol> 2. Herstellung von gemischten Zellpopulation <ol><li> Ohne schädliche Hirngewebe mit einer Schere oder einem Messer, übertragen das Gewebe mit einer 10 ml Pipette in ein steriles 50 ml konischen Röhrchen. </li><li> Zentrifuge bei 2500 RCF konischen für 5 min bei 4 ° C </li><li> Absaugen des Überstandes. Fügen Sie 4-5 ml frischem L-15-Medien (Leibowitz L-15 + 0,1% BSA + 1% Pen / Strep). </li><li> Pipette Gewebe nach oben und unten 10 Mal mit einem sterilen 10 ml Pipette. </li><li> Legen Sie eine Zelle Sieb (100 um Poren) auf ein frisches 50 ml konischen Röhrchen. Pipette Gewebe nach oben und unten einmal mit einer sterilen 5 ml Pipette und mit der Pipette bündig mit der Zelle Sieb, verzichtet das Material durch die Zelle Sieb in das konische Röhre. </li><li> Spülen Sie die Zelle Sieb mit 4-5 ml frischem L-15-Medien (Leibowitz L-15 + 0,1% BSA + 1% Pen / Strep). </li><li> Zentrifugieren konisch mit gespannten Zellen bei 2.500 RCF für 5 min bei 4 ° C </li></ol> 3. Plating und Wartung von Mixed Gliazelle Kulturen <ol><li> Für jedes Jungtier Gehirn verarbeitet werden, bereiten 1 sterile T-75-Kolben durch Zugabe von 12 ml Kulturmedium (DMEM + 10% FBS + 1% Penicillin / Streptomycin) in jeden Kolben. </li><li> Den Überstand von den pelletierten Zellen und fügen 5-6 ml Kulturmedium zu dem Zellpellet. Und Abpipettieren 10 Mal mit einer 10 ml Pipette. </li><li> Übertragen Sie ein gleiches Volumen an Zellsuspension zu jedem T-75-Kolben. </li><li> Inkubieren Flaschen in einem 5% CO 2-Inkubator bei 37 ° C für insgesamt 1-3 Wochen. </li><li> Flaschen sollten zunächst für 5 Tage bebrütet werden, und am fünften Tag, ersetzen Sie die Kulturmedien in jeder Flasche mit 12 ml frisches Medium und zurück in den Inkubator. Dann, alle 3 Tage, ersetzen Sie die konditionierten Medien in jeder Flasche mit 12 ml frisches Medium bis zur Konfluenz zu erreichen. Diese werden muss tunne sehr sorgfältig ohne den Boden berührt der Kolben in dem die Zellen anhaften. </li></ol> 4. Isolierung und Beschichtung von primären Mikroglia <ol><li> Nach gemischten Glia-Kulturen vollständig konfluent sind, entfernen Flaschen aus dem Inkubator und decken Kolben Kappen mit Parafilm den Gasaustausch mit der Umgebungsluft zu verhindern. </li><li> Schüttelkolben bei 100 rps (Lab Companion SI-600, Jeio Tech) für 1 Stunde bei 37 ° C </li><li> Sammeln Medien aus allen Kolben mit einer 10 ml Pipette, ohne dass der Astrozyten Schicht auf der Kolbenoberfläche. Legen Sie die Medien in 50 ml konischen Röhrchen. Fügen Sie frisches Medium zu den Kolben und Flaschen zurück in den Inkubator. </li><li> Zentrifuge konische Röhrchen bei 2500 RCF für 5 min bei 4 ° C </li><li> Absaugen des Überstandes aus allen konischen Röhrchen. Die Zellpellets sind hochreine Mikroglia-Zellen. Resuspendieren Pellets in 1 ml der Mikroglia Plattierung (DMEM + 10% FES + 1% Penicillin / Streptomycin). </li><li> Zählen Sie die Zelldichte in den Medien mit resuspendierteneinem Hämocytometer. </li><li> Setzen Sie einen passenden Volumen von Mikroglia Plattierungsmedien um ein 2 x 10e5 Zellen pro ml Dichte zu erreichen. Platte als geeignet für experimentelle Analyse. </li><li> Erlauben Sie Mikroglia über Nacht zu befestigen. </li></ol> 5. Repräsentative Ergebnisse Die oben beschriebenen Protokoll Ergebnisse in hoher Reinheit primären Kulturen Mikroglia. Durch immunhistochemische für einen Makrophagen / Mikroglia-Zell-spezifische Marker (Iba1) bestimmt, sind vernickelt Mikroglia Kulturen&gt; 90% (Abbildung 2). Zusätzlich, Anfärbung von Astrozyten, Oligodendrozyten und Neuronen-spezifische Marker Zelle zeigt minimale Kontamination (Abbildung 2). Nach der Gründung gemischten Glia-Zellkulturen, können Mikroglia durch Schütteln bis zu 4 aufeinander folgenden Zeitpunkten isoliert werden. Der anfängliche Isolation Mikroglia wird nachgeben etwa 2,2 x 106 Zellen / ml oder etwa 9 Millionen Zellen pro 10 Flaschen und dem erwarteten Ertrag verringert sich mit jedem weiteren shake. Mikrogliazellen mit dieser Methode isoliert wurden erfolgreich zur Phagozytose Fähigkeit, Funktion wie die Produktion von Stickoxid, und neuronale Toxizität (Abbildung 3) 13 bis 14 untersuchen verwendet. <img alt="1" src="/files/ftp_upload/3814/3814fig1.jpg" /> 1. Übersicht des Protokolls zur Herstellung von gemischten Kulturen Gliazellen und Isolierung der primären Mikroglia. <img alt="2" src="/files/ftp_upload/3814/3814fig2.jpg" /> Abbildung 2. Isolierung von hochreiner Mikroglia als durch immunhistochemische Analyse unter Verwendung von Mikroglia (Iba-1, rot), Astrozyten (GFAP, grün), Neuron (NeuN, rot) und Oligodendrozyten nachgewiesen (CC1, rot) spezifische Marker. Die Färbung für DNA von Zellen in Kultur durch DAPI (blau) wird ebenfalls angezeigt. <img alt="Abbildung 3" src="/files/ftp_upload/3814/3814fig3.jpg" /> Abbildung 3. Mikroglia isoliert in der Methode beschrieBett in einer Reihe von Tests, einschließlich verwendet worden, aber nicht beschränkt auf, Phagozytose Assays,-Assays und Neurotoxizität beschränkt. In diesen Studien haben wir gefunden, dass Mikroglia (Iba-1-positiven, grün), bei Bestrahlung mit steuern (A) oder LPS (B) behandelten Medien, die Erhöhung des Phagozytose von fluoreszenzmarkierten Latexkügelchen (rot), die quantifiziert werden kann ( C). Ferner kann LPS (1ng/ml) Behandlung zu erhöhen die Produktion von Stickoxid in Mikroglia (D). Schließlich sowohl über Transwell-Einsatz-getrennt oder direkt Mikroglia-Neuron-Kulturen, haben wir festgestellt, dass Mikroglia mit LPS kann neuronalen Zelltod induzieren wie von Lactatdehydrogenase (LDH) Freisetzung (E, F) gemessen.

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A., Pajoohesh-Ganji, A., Byrnes, K. R., Faden, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+metabotropic+glutamate+receptor+5+modulates+microglial+reactivity+and+neurotoxicity+by+inhibiting+NADPH+oxidase.">Activation of metabotropic glutamate receptor 5 modulates microglial reactivity and neurotoxicity by inhibiting NADPH oxidase.</a> J. Biol. Chem. 284, 15629-15639 (2009).</li></ol>

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Während dieses Protokoll routinemäßig benutzt wird, um reines und gesundes Mikroglia Experimente für die Forschung zu produzieren, wird sorgfältige Prüfung der technischen Aspekte bei der kritischen Teile des Verfahrens die Variabilität zu minimieren in dem isolierten Mikroglia. Erstens, während der Präparation von Hirngewebe aus neugeborenen Ratten Welpen, arbeitet in einer zeitgemäßen Weise ist notwendig, um hypoxische und ischämische Schädigung des Gewebes zu minimieren. Es ist jedoch auch wichtig, um di...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> 60 mm x 15 mm Petrischalen </td><td> Marke Fisher </td><td> 0875713A </td><td></td></tr><tr><td> Sharp Präparierscheren </td><td> Fine Science Tools </td><td> 14094-11 </td><td></td></tr><tr><td> Dumont # 7b Pinzette-Standard-Spitzen, gebogen, 11cm </td><td> Fine Science Tools </td><td> 11270-20 </td><td></td></tr><tr><td> Dumont Nr. 5 Zangen-Standard-Spitzen, gerade, 11 cm </td><td> Fine Science Tools </td><td> 11251-10 </td><td></td></tr><tr><td> 50 ml konischen Zentrifugenröhrchen </td><td> VWR </td><td> 89039-656 </td><td></td></tr><tr><td> 5 ml serologischen Pipetten </td><td> Grenier Bio One </td><td> 606180 </td><td></td></tr><tr><td> 10 ml serologischen Pipetten </td><td> Grenier Bio One </td><td> 607180 </td><td></td></tr><tr><td> 100 um sterile Nylon Zellsieb </td><td> Falke </td><td> 35-2360 </td><td></td></tr><tr><td> 75 cm 2 Zellkulturflaschen </td><td> Corning </td><td> 430641 </td><td></td></tr><tr><td> Dulbeccos Minimal Essential Medium (DMEM) </td><td> Gibco (Invitrogen) </td><td> 31053-028 </td><td></td></tr><tr><td> Leibovitz L-15-Medium </td><td> Gibco (Invitrogen) </td><td> 11415064 </td><td></td></tr><tr><td> Fötales Rinderserum </td><td> Gibco (Invitrogen) </td><td> 16000-036 </td><td></td></tr><tr><td> Fetale Pferdeserum </td><td> Fischer </td><td> SH3007402 </td><td></td></tr><tr><td> Penicillin-Streptomycin </td><td> Gibco (Invitrogen) </td><td> 15140163 </td><td></td></tr><tr><td> 100% Ethanol </td><td> Die Warner Graham Company </td><td> 64-17-5 </td><td></td></tr><tr><td> Phosphat-gepufferte Kochsalzlösung, 10X, pH 7,4 </td><td> Quality Biological, Inc. </td><td> 119-069-131 </td><td> 1X in sterilem, destilliertem Wasser </td></tr><tr><td> Biohazard Taschen </td><td> VWR </td><td> 14220-028 </td><td></td></tr><tr><td> Hämocytometer </td><td> Hausser Scientific </td><td> 1492 </td><td></td></tr><tr><td> 6-well-Zellkulturplatten mit Cellbind Oberfläche </td><td> Corning </td><td> 3335 </td><td></td></tr></tbody></table>

primary microglia isolation from mixed glial cell cultures of neonatal rat brain tissue

Mikroglia-Konto für ca. 12% der gesamten Zellpopulation im Gehirn von Säugetieren. Während Neuronen und Astrozyten die wichtigsten Zelltypen des Nervensystems angesehen werden, spielen eine bedeutende Rolle Mikroglia im normalen Gehirn-Physiologie durch die Überwachung Gewebe für Schmutz und Krankheitserregern und Aufrechterhaltung der Homöostase im Parenchym über Phagozytoseaktivität 1,2. Mikroglia sind während einer Reihe von Verletzungen und Krankheiten, einschließlich neurodegenerativer Erkrankungen, traumatische Hirnverletzung, und das Nervensystem Infektion 3 aktiviert. Unter diesen aktivierenden Bedingungen, erhöhen die Mikroglia Phagozytenaktivität, unterzogen morpohological und proliferative Veränderungen und aktiv sezernieren reaktiven Sauerstoff und Stickstoff Arten, entzündungsfördernde Zytokine und Chemokine, oft Aktivieren eines parakrine oder autokrine Schleife 4-6. Da diese Mikrogliazellen Antworten auf Krankheitsentstehung beitragen, neurologischen Erkrankungen, gezielter Forschung auf microglia gerechtfertigt ist. Aufgrund der zellulären Heterogenität des Gehirns, ist es technisch schwierig, eine ausreichende Mikroglia Probenmaterial mit hoher Reinheit zu erhalten während der in vivo-Experimente. Aktuelle Forschungen über die neuroprotektiven und neurotoxischen Funktionen von Mikroglia erfordern eine technische Routine-Methode, um konsequent zu generieren rein und gesund Mikroglia mit ausreichenden Ertrag für das Studium. Wir präsentieren, in Text und Video, ein Protokoll zur reinen primären Mikroglia aus gemischten Glia Kulturen zu isolieren für eine Vielzahl von nachgelagerten Anwendungen. Kurz gesagt, nutzt diese Technik dissoziierten Hirngewebe von neugeborenen Ratten Welpen zu gemischten Glia-Zellkulturen zu produzieren. Nachdem die gemischten Kulturen glialen Konfluenz erreichen, werden die primären Mikroglia mechanisch von der Kultur durch eine kurze Dauer Schütteln isoliert. Die Mikroglia sind dann in hoher Reinheit für die experimentelle Untersuchung überzogen. Das Prinzip und das Protokoll dieser Methodik wurdenin der Literatur beschrieben 7,8. Darüber hinaus werden alternative Methoden zur primären Mikroglia isolieren gut beschrieben 12.09. Homogenisierte Hirngewebe kann durch Dichtegradientenzentrifugation getrennt werden, um primäre Mikroglia 12 ergibt. Jedoch ist die Zentrifugation von mittlerer Länge (45 Min.) und kann eine Zellschädigung und Aktivierung sowie verursachen angereicherten Mikroglia und anderen zellulären Populationen führen. Ein anderes Protokoll wurde verwendet, um primäre Mikroglia in einer Vielzahl von Organismen zu isolieren durch längeres (16 Stunden), während Schütteln in Kultur 9-11. Nach dem Schütteln wird das Medium Überstand zentrifugiert, um Mikroglia zu isolieren. Diese längere Zwei-Schritt-Isolierung Verfahren kann auch stören Mikroglia-Funktion und Aktivierung. Wir nutzen vor allem die folgenden Mikroglia Isolation Protokoll in unserem Labor für eine Reihe von Gründen: (1) primäre Mikroglia in vivo simulieren Biologie treuer als verewigt Nagetier Mikroglia-Zelllinien, (2) Nominalen mechanischen Störungen minimiert potenzielle Zellfunktionsstörungen oder Aktivierung, und (3) ausreichende Ausbeute ohne Durchgang der gemischten Gliazellen Kulturen erhalten werden. Es ist wichtig zu beachten, dass dieses Protokoll Hirngewebe von Neugeborenen verwendet Rattenjungen Mikroglia isolieren und dass die Verwendung von älteren Ratten zu isolieren Mikroglia wesentlich beeinflussen können die Ausbeute, Aktivierungsstatus und funktionellen Eigenschaften von isolierten Mikroglia. Es gibt Hinweise darauf, dass Altern mit Mikroglia Dysfunktion, erhöhte Neuroinflammation und neurodegenerativen Erkrankungen verknüpft ist, so haben frühere Studien Mikroglia ex vivo Erwachsenen verwendet werden, um ein besseres Verständnis der Rolle der Mikroglia bei neurodegenerativen Erkrankungen, wo Altern ist wichtiger Parameter. Jedoch können ex vivo Mikroglia in Kultur nicht für längere Zeit aufbewahrt werden. Daher, während dieses Protokoll verlängert die Lebensdauer der primären Mikroglia in Kultur ist darauf hinzuweisen, dass die Mikroglia d verhaltenifferently aus adulten Mikroglia und in-vitro-Studien sollte sorgfältig abgewogen werden, wenn zu einem In-vivo-Einstellung übersetzt.

Watch this Scientific Journal Video about Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue at JoVE.com

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

Isolieren primären Mikroglia aus der zellulären Heterogenität des Gehirns ist wichtig, um deren Rolle bei physiologischen und pathologischen Bedingungen zu untersuchen. Dieses Protokoll beschreibt eine mechanische Isolation und gemischten Zellkultur-Technik, die eine hohe Ausbeute und hoher Reinheit liefert, lebensfähigen primären Mikrogliazellen für<em&gt; In-vitro-</em&gt; Studien-und Downstream-Anwendungen.

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 ...

primary-microglia-isolation-from-mixed-glial-cell-cultures-neonatal

Research

JoVE Journal

Immunology and Infection

39.1K Aufrufe.  Uniformed Services University. Isolieren primären Mikroglia aus der zellulären Heterogenität des Gehirns ist wichtig, um deren Rolle bei physiologischen und pathologischen Bedingungen zu untersuchen. Dieses Protokoll beschreibt eine mechanische Isolation und gemischten Zellkultur-Technik, die eine hohe Ausbeute und hoher Reinheit liefert, lebensfähigen primären Mikrogliazellen für In-vitro- Studien-und Downstream-Anwendungen.

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

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

Eine einfache und effiziente Methode, um Makrophagen aus Mixed primären Kulturen von adulten Leber Zellen zu isolieren

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Forschung

Immunologie und Infektion

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)1. The life cycle of HPV is tightly linked to the differentiation of squamous epithelium2. Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production3,4,5. In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras6 and modified by Kopan et al.7, the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies8. Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as well as adenoviruses, parvoviruses, and poxviruses9. Organotypic raft cultures can thus be adapted to examine viral pathogenesis, and are the only means to test novel antiviral agents for those viruses that are not cultivable in permanent cell lines.

An in vitro method to mimic in vivo epithelial differentiation is described. Many viruses target epithelial cells as part of their viral life cycle, and this method provides a means of examining virus:host interactions that more closely resembles that which occurs in vivo. This technique can be used with primary keratinocytes, established cell lines, as well as normal or diseased biopsy tissue.

Generierung von Organotypische Floßkulturen aus primären humanen Keratinozyten

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

Culturing Microglia from the Neonatal and Adult 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 pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

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 ...

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 ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

Isolierung und intravenöse Injektion von Maus-Knochenmark stammenden Monozyten

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of puberty - more than ten years after the establishment of systemic immune tolerance. Spermatogenic cells express a number of proteins that may be seen as non-self by the immune system. The testis must then be able to establish tolerance to these neo-antigens on the one hand but still be able to protect itself from infections and tumor development on the other hand. Therefore the testis is one of a few immune privileged sites in the body that tolerate foreign antigens without evoking a detrimental inflammatory immune response. Sertoli cells play a key role for the maintenance of this immune privileged environment of the testis and also prolong survival of cotransplanted cells in a foreign environment. Therefore primary Sertoli cells are an important tool for studying the immune privilege of the testis that cannot be easily replaced by established cell lines or other cellular models. Here we present a detailed and comprehensive protocol for the isolation of Sertoli cells - and peritubular cells if desired - from rat testes within a single day.

Primary Sertoli cells are required for studying testis-immune privilege, signal transduction during inflammation or infection, and utilization of their immunoprotective properties. Here we describe an enzyme-based protocol for the isolation of highly purified primary Sertoli cells and peritubular cells from rat testes.

Isolierung von Sertoli-Zellen und peritubulären Zellen aus Ratte Hoden

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Isolation und Analyse über Durchflusszytometrie von Immunzellen aus dem ischämischen Gehirn der Maus

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

Die Induktion einer entzündlichen Reaktion in der Primär Hepatozyten-Kulturen von Mäusen

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After First Colostrum Feeding

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression of proteins known to respond to metabolic stress was measured in brain-nuclei isolates using Western blotting. This was done to assess whether metabolic stress-induced nutrient insufficiency in the body triggered neuronal stress. We have previously demonstrated that nutrient insufficiency in neonates elicits metabolic stress in the gut. Furthermore, colostrum oxytocin modulates cellular stress response, inflammation, and autophagy markers in newborn rat gut villi prior to and after first feed. Signaling protein markers associated with the endoplasmic reticulum stress [ER chaperone binding immunoglobulin protein (BiP), eukaryotic translation initiation factor 2A (eIF2a), and eIF2a kinase protein kinase R (p-PKR)], as well as two inflammation-signaling proteins [nuclear factor-&#954;B (NF-kB) and inhibitor &#954;B (IkB)], were measured in newborn brain nuclei [nucleus of the solitary tract (NTS), paraventricular nucleus (PVN), supra-optic nucleus (SON), cortex (CX), striatum nuclei (STR), and medial preoptic nucleus (MPO)] before the first feed (unprimed by colostrum) and after the start of nursing (primed by colostrum). Expression of BiP/GRP78 and p-eIF2a were upregulated in unprimed and downregulated in primed NTS tissue. NF-kB was retained (high) in the CX, STR, and MPO cytoplasm, whereas NF-kB was lower and unchanged in NTS, PVN, and SON in both conditions. The collective BiP and p-eIF2 findings are consistent with a stress response. eIf2a was phosphorylated by dsRNA dependent kinase (p-PKR) in the SON, CX, STR, and MPO. However, in the NTS (and to a lesser extent in PVN), eIf2a was phosphorylated by another kinase, general control nonderepressible-2 kinase (GCN2). The stress-modulating mechanisms previously observed in newborn gut enterocytes appear to be mirrored in some OTR-rich brain regions. The NTS and PVN may utilize a different phosphorylation mechanism (under nutrient deficiency) from other regions and be refractory to the impact of nutrient insufficiency. Collectively, this data suggests that brain responses to nutrient insufficiency stress are offset by signaling from colostrum-primed enterocytes.

Here, we present a protocol to isolate brain nuclei in the neonatal rat brain in conjunction with first colostrum feeding. This technique allows the study of nutrient insufficiency stress in the brain as modulated by enterocyte signaling.

Bewertung von zellulären Stresses und Entzündung im diskreten Oxytocin-sezernierenden Gehirn Kerne bei Neugeborenen Ratten vor und nach der ersten Fütterung Kolostrum

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression ...

Concomitant Isolation of Primary Astrocytes and Microglia for Protozoa Parasite Infection

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central nervous system (CNS). The increasing evidences of their involvement in the control of infectious diseases justify the emerging interest in the improvement of methodologies to isolate primary astrocytes and microglia in order to evaluate their responses to infections that affect the CNS. Considering the impact of Trypanosoma cruzi (T. cruzi) and Toxoplasma gondii (T. gondii) infection in the CNS, here we provide a method to extract, maintain, dissociate and infect murine astrocytes and microglia cells with protozoa parasites. Extracted cells from newborn cortices are maintained in vitro for 14 days with periodic differential media replacement. Astrocytes and microglia are obtained from the same extraction protocol by mechanical dissociation. After phenotyping by flow cytometry, cells are infected with protozoa parasites. The infection rate is determined by fluorescence microscopy at different time points, thus enabling the evaluation of differential ability of glial cells to control protozoan invasion and replication. These techniques represent simple, cheap and efficient methods to study the responses of astrocytes and microglia to infections, opening the field for further neuroimmunology analysis.

The overall goal of this protocol is to instruct how to extract, maintain, and dissociate murine astrocyte and microglia cells from the central nervous system, followed by infection with protozoa parasites.

Gleichzeitige Isolierung von Primärastrozyten und Mikroglia bei Protozoenparasiten-Infektion

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central ...