Diese Arbeit wurde durch Grant NS64571 aus dem NINDS (CLH) unterstützt wird, durch eine frühzeitige Career Development Award von der Mayo Clinic (CLH), und durch ein großzügiges Geschenk von Donald und Frances Herdrich (CLH). Wir möchten die Mayo Clinic Durchflusszytometrie Core for Unterstützung danken.

1. Intrakranielle Virus Injektion: Das folgende Verfahren wurde geändert und weitgehend durch unser Labor und Kollegen genutzt. Kurz gesagt, intrakranieller Injektion des Daniel Stamm murinen Enzephalomyelitis Theiler ist Virus (TMEV) oder schein-Infektion (1, 10) ist auf junge Mäuse (vorzugsweise 5-6 Wochen alt) durchgeführt, um Gehirn infiltrieren Leukozyten (Bils) zu entlocken. Bitte beachten Sie, dass die Ergebnisse wird zwischen den Daniel Stamm, die Bean-Stamm, und der GDVII Belastung unterscheiden. Für die Zwecke der Ernte Bils, erhalten Mäusen 2x10 5 PFU von TMEV und sind für 24 Stunden infiziert. <ol><li> Bringen Sie die Injektionsnadel (27 Gauge, ¼ in, Kendall), eine automatische 1 ml Hamilton-Spritze und auf 10 ul des Virus liefern. </li><li> Erstellen Sie TMEV in 10 ul DMEM in die Spritze mit besonderer Aufmerksamkeit, um Luftblasen. Gegebenenfalls erhalten sham infizierten Mäusen 10 ul virenfrei DMEM. </li><li> Unmittelbar vor der Injektion, pour etwa 1 mL von Isofluran in einer Glasglocke, dass ein Drahtgitter mit Baumwolle Substrat unterhalb enthält. </li><li> Legen Sie einfach direkt auf Mäuse Drahtgitter in der Glocke, bis sie leicht narkotisiert werden, unempfindlich gegen toe-kneifen und immobilisiert durch die Inhalations-Anästhetika, ca. 10-15 Sekunden. Mäuse sollten nicht in direkten Kontakt mit Isofluran kommen, als Substanz kann irritierend sein und ätzend. </li><li> Während der Narkose, bereiten Mäuse für die Injektion von schnellen Auffinden geeigneter Koordinaten im frontalen Kortex Region auf dem Schädel mit Rasier-und Farbgebung. Die allgemeine Lage für die Injektion ist 1 mm anterior zu Bregma und 1 mm seitlich der Sagittalnaht auf der rechten Seite (Abbildung 1). Während stereotaktische Injektion kann sinnvoll sein für einige experimentelle Situationen haben wir festgestellt, dass freie Hand Injektion ohne Rasieren oder Einfärben geeignet für die Mehrzahl der Experimente ist. Das Fell mit der Nadelspitze an beiden Achsen eignet sich für die Injektion. </li><li> Um eine Injektion zu machen, richten Sie die Nadel senkrecht auf den Schädel, und drücken durch den Knochen auf ca. 3 mm Tiefe. </li><li> Express-Virus in das Gehirn, indem Sie die Hamilton-Spritze gedrückt und wartet 2 Sekunden für das Virus auf das Gehirn vor Aberkennung geben. </li><li> Entfernen Sie vorsichtig die Nadel und die Maus in einem sauberen, trockenen Käfig. Mäuse aus der Narkose erwachen und schnell wieder normale Funktion innerhalb weniger Minuten. Give gebührende Aufmerksamkeit zu Mäusen, die anormale Symptome aufweisen, wie zB starke Blutungen an der Injektionsstelle, wie Euthanasie der Tiere angemessen sein. Richtiger Umgang mit Tier 6 und die Einhaltung der National Institutes of Health and Institutional Animal Care und Verwenden Ausschuss Richtlinien müssen befolgt werden. </li></ol> 2. Brain-infiltrierenden Leukozyten Zellpräparation: <ol><li> Bereiten Rundboden-Oak Ridge Zentrifugenröhrchen mit einer Kapazität von 30 ml mit 10 ml RPMI 1640, 9 ml Percoll und 1 ml 10X PBS und Rohre sollten bei RT auf der Bank sitzen während des Gehirns Löschvorgang halten kann. </li><li> Bringen Ficoll-Paque Plus Raumtemperatur (RT). </li><li> Euthanize Tier durch veränderte Isofluran Überdosierung 5 und schnell zu entfernen, das Gehirn mit einem Edelstahl-Gewebe Spachtel und in einem 15 ml konischen Röhrchen mit 5 ml RPMI auf Eis. Unter bestimmten Umständen kann PBS-Perfusion angemessen zu entfernen zirkulierenden peripheren Blutzellen aus dem Bils Vorbereitung. Mäuse sollten vollständig und ordnungsgemäß betäubt vor PBS-Perfusion. </li><li> Transfer-Hirn-und RPMI-Lösung zu einem 7 ml Glas Pyrex Marke Tenbroeck Gewebe Mühle und sanft mit 10-15 Schlägen zu homogenisieren. </li><li> Pipet weitere 5 ml RPMI in den Homogenisator und Pipette nach oben und unten zweimal. Transfer Hirnhomogenat (ca. 10 ml) auf den vorbereiteten Rundboden-Röhrchen und vorsichtig umdrehen 2-3 mal zu mischen. </li><li> Spin Hirnhomogenat bei 7800g ave für 30 min bei RT in einer F0360 Festwinkelrotor in einem Beckman Allegra X-22R klinischen Zentrifuge. </li><li> Nach dem Zentrifugieren zu entfernen Myelin Trümmer, schwebte auf der Oberseite der Steigung hat. Sammeln Sie die Leukozyten-Schicht, die oberhalb der roten Blutkörperchen Pellet (Abbildung 2) schwebt. </li><li> Der Stamm Leukozyten Schicht durch ein 40 um Zellsieb in einen 50 ml konisch. Bringen Lösung bis zu 50 ml Volumen mit RMPI. </li><li> Spin Röhrchen mit verdünnten Zellsuspension in einer klinischen Zentrifuge bei 1500 rpm (600g ave) für 5 min bei RT. </li><li> Den Überstand aspirieren und sammeln das Zellpellet. Das Pellet in 1 ml FACS-Puffer (1% Rinderserumalbumin, 0,02% Natriumazid in Calcium-und Magnesium-freiem PBS) und übertragen Sie die Zellsuspension 5 ml Rundboden FACS-Röhrchen. </li><li> Sorgfältig Unterlage Aufhängung mit 1 ml Ficoll-Paque Plus. </li><li> Spin Röhrchen bei 2500 rpm (1400 gab) in klinischen Zentrifuge für 25 min bei RT ohne Bremse. </li><li> Entfernen weißen, flauschigen Schicht an der Oberfläche (Abb. 2) mit 1 ml Pipette und Übertragung auf neue 5 ml FACS-Röhrchen. Wash cEllen mit 4 ml FACS-Puffer. </li><li> Spin Röhrchen bei 1500 rpm (600g ave) für 5 min bei RT in klinischen Zentrifuge zur Pellet-Zellen. </li><li> Saugen Sie den Überstand und sammeln Zellpellet. Resuspendieren Leukozyten Pellet in geeigneten Puffer und halten auf dem Eis in neuen FACS-Röhrchen. </li><li> Zählen von Zellen, beurteilen die Lebensfähigkeit der Zellen durch Trypanblau-Ausschluss und zu analysieren, mittels Durchflusszytometrie. Im Allgemeinen können die Ermittler erwarten, ca. 5 x 10 5 Zellen / Tier zu erwerben. </li></ol> 3. Durchflusszytometrische Immunphänotypisierung <ol><li> Nach Isolierung und Zählung der Leukozyten, Spin die Zellen für 3 Minuten bei 1500 rpm (600 gab) in einer klinischen Zentrifuge. </li><li> Das Pellet in Blocking-Puffer mit folgendem Inhalt: FACS-Puffer, Überstand aus 2.4G2 Hybridom (Fc block; anti-CD16/32) und fötalem Rinderserum bei einem Verhältnis von 10.05.01 und inkubieren für 30 Minuten bei 4 ° C . </li><li> Bereiten Sie und fügen konjugierte Antikörper gegen extrazelluläre Antigene, um die blockierten Zellen bei einer Konzentration von 1:200 und Inkubation für 30 Minuten bei 4 ° C im Dunkeln. Stain und inkubieren Zellen in 200 ul in einem 96-Loch-V-Bodenplatte. Entsprechende Kontrollen sind ungefärbte Zellen und Fluorochrom Entschädigung Proben, wie nötig. </li><li> Drehen Sie die gefärbten Zellen bei RT für 3 Minuten bei 1500 rpm (600 gab) in einer klinischen Zentrifuge mit einem Rotor geeignet für 96-Well-Platten. </li><li> Den Überstand aspirieren und waschen Sie die Zellen mit 200 ul FACS-Puffer durch vorsichtiges Auf-und Abpipettieren 3 mal. </li><li> Wiederholen Sie die obigen Waschtechnik zweimal. </li><li> Nach dem Waschen die Zellen in 2% Paraformaldehyd und Transfer zum FACS-Röhrchen. Fixation ist für den durchflusszytometrischen Analyse von Zellen des Biosafety Level 2 Reagenzien infiziert erforderlich. </li><li> Führen Sie den fixierten Zellen auf einer BD FACS Calibur nach einer modifizierten Durchflusszytometrie Methode 8 und analysieren Dateien offline mit FlowJo, WinMDI oder anderen verfügbaren Durchflusszytometrie-Analyse-Software. </li><li> Die Analyse der 50-100,000 Ereignisse pro Probe ist in der Regel für eine angemessene Immunphänotypisierung erforderlich. </li></ol> Direkt konjugierte Antikörper in diesem Experiment verwendet: CD45 wurde mit Klon 30-F11 erkannt. Ly6C / G wurde mit Klon Gr1, RB6-8C5 erkannt. CD11b wurde mit Klon M1/70 erkannt. F4/80 wurde mit Klon BM8 erkannt. 4. Repräsentative Ergebnisse: Wir zeigen Zelle Phänotypisierung Ergebnisse für Maus Bils 24 Stunden nach der Infektion (Abbildung 3). Leukozyten mit PerCP-konjugiertem anti-Maus CD45 gefärbt wurden, um Immunzellen erkennen, PE-konjugierten Anti-Maus Ly6C / G zu erkennen inflammatorischen Monozyten, APC-konjugierten Anti-Maus-CD11b und APC-konjugierten Anti-Maus F4/80, um Zellen zu erkennen von Monozyten Linie. Die Analyse wurde mit einem BD FACS Calibur Gerät durchgeführt. <img src="/files/ftp_upload/2747/2747fig1.jpg" alt="Abbildung 1"/> Abbildung 1. Anatomische Lokalisation der Injektionsstelle. Die Injektionsstelle ist 1 mm anterior zu Bregma und 1 mm seitlich der Sagittalnaht auf der rechten Seite befindet. <img src="/files/ftp_upload/2747/2747fig2.jpg" alt="Abbildung 2"/> Abbildung 2. Illustration von Gradienten Trennung von Leukozyten und Bils. Leukozyten werden zunächst direkt unterhalb des Myelin Trümmer Schicht und vor der RBC Pellet im Percoll-Gradienten gesammelt. Der weiße, flauschige Schicht (Bils) an der Schnittstelle von der Ficoll-Gradienten gesammelt. <img src="/files/ftp_upload/2747/2747fig3.jpg" alt="Abbildung 3"/> Abbildung 3. Immunophänotyp von Gehirn-infilatrating Leukozyten bei 24 Stunden nach der Infektion. Maus Leukozyten wurden aus dem Gehirn durch Differential Dichtezentrifugation von Gewebehomogenaten von Tieren bei 24 Stunden nach der intrakraniellen Infektion vorbereitet isoliert. Die Zellen wurden mit Fluoreszenz-konjugierten Antikörper gegen Maus CD45 gefärbt, Ly6C / G, CD11b und F4/80. Initial-Gating wurde durch Auftragen CD45, einem Marker für Immunzellen gegen Vorwärtsstreuung (FSC), ein Indikator für die Größe der Zellen (C) durchgeführt. Die CD45 hallo (A, D), CD45 lo (B, E) und CD45 neg (F, G) Populationen wurden dann für die relative Expression der Monozyten und Makrophagen Marker Ly6C / G, F4/80 (A, B analysiert , F) und CD11b (D, E, G). Ly6C / G + F4/80 + CD11b + inflammatorischen Monozyten wurden fast ausschließlich in der CD45 hallo Bevölkerung (A, D), im Einklang mit der Infiltration dieser Zellen aus der Peripherie. Im Gegensatz dazu waren Ly6C/G-F4/80 lo CD11b + Makrophagen fast ausschließlich in der CD45 lo (B, E) und CD45 neg (G) Populationen, im Einklang mit einem Wohnsitz Phänotyp gefunden. In zahlreichen Versuchen haben wir noch nie CD45 hallo Zellen oder Ly6C / G + F4/80 + CD11b + inflammatorischen Monozyten in das Gehirn der infizierten oder schein-Infektion beobachtetTed Mäusen (Daten nicht gezeigt).

<ol>
	<li>Buenz, E. J., Howe, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picornaviruses+and+cell+death.">Picornaviruses and cell death.</a> Trends Microbiol. 14, 28-36 (2006).</li><li>Buenz, E. J., Limberg, P. J., Howe, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+3-parameter+flow+cytometry-based+cell+death+assay.">A high-throughput 3-parameter flow cytometry-based cell death assay.</a> Cytometry A. 71, 170-173 (2007).</li><li>Deb, C., Lafrance-Corey, R. G., Zoecklein, L., Papke, L., Rodriguez, M., Howe, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demyelinated+axons+and+motor+function+are+protected+by+genetic+deletion+of+perforin+in+a+mouse+model+of+multiple+sclerosis.">Demyelinated axons and motor function are protected by genetic deletion of perforin in a mouse model of multiple sclerosis.</a> J. Neuropath. Exp. Neurol. 68, 1037-1048 (2009).</li><li>Deb, C., Howe, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NKG2D+contributes+to+efficient+clearance+of+picornavirus+from+the+acutely+infected+murine+brain.">NKG2D contributes to efficient clearance of picornavirus from the acutely infected murine brain.</a> J. Neurovirol. 14, 261-266 (2008).</li><li>Donovan, J., &amp;amp, P. . B. r. o. w. n., , P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Euthanasia.">Euthanasia.</a> Current Protocols in Neuroscience. , A.4H.1-A.4H.4 (2005).</li><li>Donovan, J., Brown, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Handling+&#38;+restraint.">Handling &#38; restraint.</a> Current Protocols in Immunology. 73, 1.3.1-1.3.6 (2006).</li><li>Donovan, J., Brown, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parenteral+injections.">Parenteral injections.</a> Current Protocols in Neuroscience. , A.4F.1-A.4F.9 (2005).</li><li>Holmes, K. L., Otten, G., Yokoyama, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+analysis+using+Becton+Dickinson+FACS+Calibur.">Flow cytometry analysis using Becton Dickinson FACS Calibur.</a> Current Protocols in Immunology. , 5.4.1-5.4.22 (2002).</li><li>Howe, C. L., Ure, D., Adelson, J. D., LaFrance-Corey, R., Johnson, A., Rodriguez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD8++T+cells+directed+against+a+viral+peptide+contribute+to+loss+of+motor+function+by+disrupting+axonal+transport+in+a+viral+model+of+fulminant+demyelination.">CD8+ T cells directed against a viral peptide contribute to loss of motor function by disrupting axonal transport in a viral model of fulminant demyelination.</a> J Neuroimmunol. 188, 13-21 (2007).</li><li>Lipton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theiler's+virus+infection+in+mice:+An+unusual+biphasic+disease+process+leading+to+demyelination.">Theiler's virus infection in mice: An unusual biphasic disease process leading to demyelination.</a> Infect Immun. 11, 1147-1155 (1975).</li></ol>

Wir verwenden routinemäßig Durchflusszytometrie, um sowohl die Qualität des Gehirns infiltriert Zellpräparation zu bestimmen und verschiedene Populationen von Immunzellen 2, 9 zu unterscheiden. Bei akuten Zeit-Punkte, ergibt unsere Bils Methode ein hoher Prozentsatz der inflammatorischen Monozyten in der CD45hi Bevölkerung sowie ein hoher Prozentsatz der Makrophagen in der CD45lo Bevölkerung. Dies deutet darauf hin, dass eine reproduzierbare Immunantwort im Gehirn robust können mit unserer Methode char...

<table border="1"><tbody><tr><th> Reagens </th><th> Firma </th><th> Katalog-Nummer </th><th> Kommentare </th></tr><tr><td> TMEV </td><td> Howe Lab </td><td> N / A </td><td></td></tr><tr><td> Daniel Stamm </td><td> Invitrogen </td><td> 21063-029 </td><td></td></tr><tr><td> Isofluran </td><td> Novaplus </td><td> NDC 0409-3292-49 </td><td></td></tr><tr><td> Rundboden-Oak Ridge Zentrifugenröhrchen </td><td> Nalgene </td><td> 3118-0030 </td><td></td></tr><tr><td> RPMI 1640 </td><td> Invitrogen </td><td> 11875-093 </td><td></td></tr><tr><td> Percoll </td><td> GE Healthcare </td><td> 17-0891-02 </td><td></td></tr><tr><td> 10X PBS </td><td> Roche </td><td> 11666789001 </td><td></td></tr><tr><td> Ficoll-Paque Plus- </td><td> GE Healthcare </td><td> 17-1440-02 </td><td></td></tr><tr><td> Trypanblau 0,4% (w / v) </td><td> Mediatech </td><td> 25 bis 900-CI </td><td></td></tr><tr><td> 15 ml konische Röhrchen </td><td> BD Falcon </td><td> 352097 </td><td></td></tr><tr><td> 7 ml Glas Pyrex Marke Tenbroeck Gewebe grinder </td><td> Fischer </td><td> 08 bis 414-10B </td><td></td></tr><tr><td> 40 um Zellsieb </td><td> BD Falcon </td><td> 352340 </td><td></td></tr><tr><td> 50 ml konischen </td><td> BD Falcon </td><td> 352070 </td><td></td></tr><tr><td> Rinderserumalbumin </td><td> Sigma </td><td> A9647 </td><td></td></tr><tr><td> Natriumazid </td><td> Sigma </td><td> S8032 </td><td></td></tr><tr><td> CMF-PBS </td><td> Mediatech </td><td> 21 bis 040-CV </td><td></td></tr><tr><td> FACS-Röhrchen </td><td> BD Falcon </td><td> 352054 </td><td></td></tr><tr><td> Rinderfötenserum </td><td> Sigma </td><td> F4135 </td><td></td></tr><tr><td> 2.4G2 Hybridom </td><td> ATCC </td><td> HB-197 </td><td></td></tr><tr><td> Costar V-Bodenplatte </td><td> Corning </td><td> 3894 </td><td></td></tr><tr><td> Allegra X-22R Zentrifuge oder eine gleichwertige </td><td> Beckman </td><td> N / A </td><td></td></tr><tr><td> 96-Well-Platte Eimer und Rotor </td><td> Beckman </td><td> S2096 </td><td></td></tr><tr><td> Festwinkelrotor </td><td> Beckman </td><td> F0360 </td><td></td></tr><tr><td> Paraformaldehyd </td><td> Sigma </td><td> P6148 </td><td></td></tr><tr><td> BD FACS Calibur </td><td> BD Biosciences </td><td> N / A </td><td></td></tr><tr><td> FlowJo 7,5 </td><td> Baum Star, Inc. </td><td> N / A </td><td></td></tr><tr><td> CD45 </td><td> BD Biosciences </td><td> 557235 </td><td> Klon: 30-F11 </td></tr><tr><td> Gr1 (Ly6C / G) </td><td> BD Biosciences </td><td> 553128 </td><td> Klon: RB6-8C5 </td></tr><tr><td> CD11b </td><td> ebiosciences </td><td> 17-0112-83 </td><td> Klon: M1/70 </td></tr><tr><td> F4/80 </td><td> ebiosciences </td><td> 17-4801-82 </td><td> Klon: BM8 </td></tr></tbody></table>

isolation of brain-infiltrating leukocytes

Wir beschreiben ein Verfahren zur Herstellung Gehirn infiltrieren Leukozyten (Bils) von Mäusen. Wir zeigen, wie man Mäuse mit Mäuse-Enzephalomyelitis Theiler ist Virus (TMEV) infizieren über eine schnelle intrakraniellen Injektionstechnik und wie man eine Leukocyten-angereicherte Population von infiltrierenden Zellen aus ganzen Gehirn zu reinigen. Kurz gesagt, werden die Mäuse mit Isofluran in einer geschlossenen Kammer und sind frei Hand injiziert mit einer Hamilton-Spritze in den frontalen Kortex. Mäuse werden dann zu verschiedenen Zeiten nach der Infektion mit Isofluran Überdosierung und ganze Gehirn getötet werden extrahiert und in RPMI homogenisiert mit einem Tenbroeck Gewebe Mahlwerk. Hirnhomogenaten werden durch eine kontinuierliche 30% Percoll-Gradienten zentrifugiert, um das Myelin und andere Zelltrümmer zu entfernen. Die Zellsuspension wird dann bei 40 um angespannt, gewaschen und zentrifugiert auf eine diskontinuierliche Ficoll-Paque Plus-Gradienten zu wählen und reinigen die Leukozyten. Die Leukozyten werden dann gewaschen und in entsprechenden Puffern für Immunphänotypisierung mittels Durchflusszytometrie. Durchflusszytometrie zeigt eine Bevölkerung von angeborenen Immunzellen in den frühen Stadien der Infektion in C57BL / 6 Mäusen. Nach 24 Stunden nach der Infektion, werden mehrere Untergruppen von Immunzellen in der Bils, mit einer angereicherten Population von Gr1 +, CD11b + und F4/80 + Zellen. Daher ist diese Methode zur Charakterisierung der Immunantwort auf eine akute Infektion in das Gehirn.

Watch this Scientific Journal Video about Isolation of Brain-infiltrating Leukocytes at JoVE.com

Isolation of Brain-infiltrating Leukocytes

Ein schnelles Verfahren zur Infiltration von Leukozyten aus dem murinen Gehirns zu erhalten, ist beschrieben. Diese Methode verwendet einen kontinuierlichen und diskontinuierlichen Percoll-Gradienten Ficollgradienten auszuwählen und zu reinigen, die Leukocyten-angereicherte Schicht. Isolierte Leukozyten kann dann durch durchflusszytometrische Messungen charakterisiert werden.

Isolierung von Brain-infiltrierenden Leukozyten

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine ...

isolation-of-brain-infiltrating-leukocytes

Research

JoVE Journal

Immunology and Infection

19.0K Aufrufe.  Mayo Clinic College of Medicine. Ein schnelles Verfahren zur Infiltration von Leukozyten aus dem murinen Gehirns zu erhalten, ist beschrieben. Diese Methode verwendet einen kontinuierlichen und diskontinuierlichen Percoll-Gradienten Ficollgradienten auszuwählen und zu reinigen, die Leukocyten-angereicherte Schicht. Isolierte Leukozyten kann dann durch durchflusszytometrische Messungen charakterisiert werden.

Serie: Isolation of Brain-infiltrating Leukocytes

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

Isolierung von Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Forschung

Immunologie und Infektion

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Isolierung von Maus Lung Dendritischen Zellen

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

Isolierung und Analyse von Brain-sequestered Leukozyten aus<em&gt; Plasmodium berghei</em&gt; ANKA-infizierte Mäuse

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

Bioenergetik und die Oxidative Burst: Protokolle für die Isolierung und Bewertung der menschlichen Leukozyten und Thrombozyten

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

Analyse der Auswirkungen von Stromazellen an der Rekrutierung von Leukozyten von Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to&#160;pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is&#160;a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

Isolation von Leukozyten aus dem menschlichen Mutter und Fötus-Schnittstelle

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

Isolierung von Leukozyten aus dem Mausgeweben an der Maternal-Fetal-Schnittstelle

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

Isolation der infiltrierenden Leukozyten aus Mäusehaut unter Verwendung enzymatischer Digest und Gradiententrennung

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Selective Harvesting of Marginating-pulmonary Leukocytes

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Evidence suggests higher cytotoxicity of natural killer cells, and a distinct pro- and anti-inflammatory profile of the MP-leukocyte population compared to circulating or splenic immunocytes. The method presented herein enables selective harvesting of MP-leukocytes by forced perfusion of the lungs in either mice or rats. In contrast to other methods used to extract lung-leukocytes, such as tissue grinding and biological degradation, this method exclusively yields leukocytes from the lung capillaries, uncontaminated with parenchymal, interstitial, and broncho-alveolar cells. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MP-leukocytes, without inducing physiological responses due to tissue processing. This unique MP leukocyte population is strategically located to identify and react towards abnormal circulating cells, as all circulating malignant cells and infected cells are detained while passing through the lung capillaries, physically interacting with endothelial cells and resident leukocytes,. Thus, selective harvesting of MP-leukocytes and their study under various conditions may advance our understanding of their biological and clinical significance, specifically with respect to controlling circulating aberrant cells and lung-related diseases.

Marginating-pulmonary leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe selective harvesting of this subpopulation of pulmonary leukocytes, by forced perfusion of the lungs in rats and mice. Marginating-pulmonary leukocytes seem critical in determining susceptibility to blood-borne and lung-related diseases.

Selektive Ernte von Marginating-Lungen-Leukozyten

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit ...

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

Selektive Ernte von Marginating-Leber-Leukozyten

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...

Methods Article

Isolierung von Brain-infiltrierenden Leukozyten