Die Autoren bedanken sich bei Frau Liana Mackiewicz für die technische Unterstützung bedanken. Diese Arbeit wurde ermöglicht durch Victorian State Government Operational Support der Infrastruktur und der australischen Regierung National Health and Medical Research Council IRIISS und Project Grant 1031212.

Ein. Infektion von Mäusen mit P. berghei-ANKA <ol><li> Defrost ein Aliquot von kryokonservierten P. berghei ANKA PRBC. </li><li> Restrain eine zerebrale Malaria-resistente BALB / c Donor-Maus (8-12 Wochen alt) mit dem two-handed Zurückhaltung Technik. Inject Maus mit 100-200 ul PRBC mit einer 1 ml Insulinspritze (28G Nadel). Routinemäßig werden 1-2 Spender-Mäusen injiziert. </li><li> Am Tag 4-5 nach der Infektion (pi) zu entfernen Donor-Maus aus dem Käfig und legen Sie sie auf einer Workstation oder einem Einweg arbeiten Pad. </li><li> Gently zurückzuhalten Maus bis zum Ende des Schwanzes und mit einer kleinen Schere schneiden Sie die Schwanzspitze (ca. 1 mm). Alternativ könnte ein kleiner Einstich mit einem Schwanz 25 G Nadel vorgenommen werden. </li><li> Geben Sie einen Tropfen Blut aus der Maus Schwanzvene am Ende einer mattierten-End-Mikroskop-Objektträger. </li><li> Legen Sie einen zweiten Schieber (Spreader) auf einem 45 ° Winkel und es zurück in den Blutstropfen. Sobald das Blut breitet sich entlang der Kante, drücken Sie den Streuer-slide evenly auf dem Objektträger um die Blutausstrich machen. Lassen Sie den Abstrich an der Luft trocknen. </li><li> Fix Blutausstrichen in 100% Methanol für 30 Sekunden und dann färben Folien in frisch hergestelltem Giemsa für 10 min. </li><li> Spülen Blutausstrich mit fließendem Wasser für 1 min, an der Luft trocknen lassen und untersuchen Objektträger unter dem Mikroskop (100x, Öl-Immersion). Aufzählen PRBC in 1.000 Erythrozyten und berechne Prozent Parasitämie. Donor Mäuse sollten Parasitämie Ebenen zwischen 2,5-5% erreichen, bevor sie für die Infektion von Versuchstieren entlüftet werden. </li><li> Blutentnahme von dem Spender Maus durch retroorbitale Blutungen unter Verwendung eines Mikro-heparinisierten Hämatokrit Kapillarrohr. Alle Versuche werden in Übereinstimmung mit den Walter &amp; Eliza Hall Institut Animal Ethics Committee Anforderungen durchgeführt. Je nach den örtlichen Tier Ethic Committee andere Anforderungen Blutungen Verfahren verwendet werden könnten. Aufzulösen erforderliche Blutmenge in RPMI-Medium mit einer Endkonzentration von 1x10 6 pRBC/0.2 ml. Bedenken Sie, dasseine normale Maus Hämatokrit ~ 6x10 9 roten Blutkörperchen / 1 ml. </li><li> Infect experimentellen C57BL / 6 Mäuse (8-12 Wochen alt) durch Einspritzen intraperitoneal (ip) 1x10 6 pRBC/0.2 ml. </li><li> Um Parasitämie Ebenen der infizierten Mäusen beobachten die Schritte 1,3-1,8. Parasitämie sollte ermittelt alle 2-3 Tage, beginnend am Tag 2 oder 3 pi </li><li> Auftreten einer schweren Malaria in C57BL / 6 Mäusen manifestieren könnte als gekrümmte Erscheinung, ruffed Fell und geringe Aktivität. Einige Mäuse können in dieser Phase zu erholen, aber Progression zu Verlust der Selbstkontrolle aufrichtenden Reflex zeigt irreversible Krankheit und Tiere müssen eingeschläfert werden. </li></ol> 2. Intrakardiale Perfusion von Mäusen und Brain Extraction <ol><li> Montieren einer manuellen Schwerkraft Perfusionssystem durch Befestigen eines 500 ml Reservoir zu einer Säule angebrachten Halter Anfang einer 60 cm Säule Ständer. Verbinden Kunststoffrohr am unteren Ende des Reservoirs und sichern eine Schlauchklemme zum Puffer zu steuern. Befestigen Sie Schlauch mit einer 23 G Nadel. Fill bis das Reservoir mit PBS (gehalten bei 22 ° C), öffnen Sie klemmen und ermöglichen Puffer durch den Schlauch laufen, um alle Luftblasen zu entfernen. </li><li> Euthanize P. berghei ANKA-infizierten Maus durch CO 2-Inhalation. Bestätigen Tod durch das Fehlen des Pedals, Schwingschleifer und respiratorischen Reaktionen. </li><li> Merken eingeschläfert Maus durch den Hinterbeinen und Vorderpfoten dorsal auf einem Styropor-Dissektion Bord in einer Plastikschale enthalten. Je nach den örtlichen Tier Ethic Committee Anforderungen Anästhesie konnte vor Beginn der intrakardialen pefusion verabreicht werden. </li><li> Wischen Bauchseite mit 70% Ethanol. </li><li> Verwenden Sie große Schere und Pinzette die Haut entlang der Mittellinie öffnen, um die Brusthöhle aussetzen. Fach und Pin Haut an den Seiten. </li><li> Halten das Brustbein mit feinen Pinzetten und schneiden das Diaphragma und auf beiden Seiten des Brustbeins Durchtrennen der Rippen. Achten Sie darauf, dass keine großen Blutgefäße schädigen. </li><li> Pin der Brustkorb von dem Brustbein lose neben dem Kopf. </li><li> Halten Ventrikel with feinen Pinzette vorsichtig incise rechten Vorhof mit feinen Schere. </li><li> Legen 23G Nadel Schwerkraft Perfusionssystem in die linke Herzkammer zur Aorta ascendens während PBS läuft. Legen Sie nur 0,5 cm der Nadelspitze. </li><li> Perfundieren Maus für 5 min oder bis Ablauf ist klar. </li><li> Unpin Maus und legen Sie sie auf ihren Bauch. Wischen Sie den Kopf mit 70% Ethanol. </li><li> Mit großen Schere, einen Schnitt oberhalb des zervikalen Rückenmarks Bereich. </li><li> Verwenden feinen Schere zu machen eine mediane caudal-rostral Schnitt beginnend an der Basis des Schädels und schälen Haut weg, um den Schädel freizulegen. </li><li> Halten den Kopf, legen Sie die Blätter eines kleinen Schere in jeder Augenhöhle und einen Schnitt zwischen den Umlaufbahnen. </li><li> Machen Sie einen Längsschnitt entlang der Pfeilnaht und sorgfältig schälen Schädel auf jeder Gehirnhälfte nach außen. </li><li> Mit einem Spatel, heben das Gehirn und legen Sie sie in einem 10 ml Zentrifugenröhrchen mit RPMI-Medium. </li></ol>3. Isolierung von Brain-sequestered Leukozyten (BSL) <ol><li> Arbeiten in einer Sicherheitswerkbank, Ort frisch Gehirns auf einem Edelstahl-Zellsieb (40-60 Maschenweite) in eine Petrischale mit 3-5 ml RPMI Zellkulturmedium geerntet. </li><li> Cut Hirngewebe in kleine Stücke schneiden. </li><li> Push kleine Stücke von Hirngewebe durch die Zelle unter Verwendung eines Kristalls Sieb Druckfläche. </li><li> Übertragen Gehirnhomogenat in ein 10 ml Röhrchen und Zentrifuge bei 250 × g für 10 min bei 4 ° C. </li><li> Das Pellet in 3 ml RPMI-Medium, das 0,05% Collagenase D und 2U/ml DNAse I. </li><li> Drehen Mischung wird 30 min bei Raumtemperatur. Zelltrümmer zu entfernen, indem das Gemisch durch einen 70 um Nylon Zellsieb und / oder durch Inkubation auf Eis für 5 min. </li><li> Lay Hirnhomogenisat auf eine 7 ml 30% Percoll Kissen und Zentrifuge bei 400 × g (ungebremst) 20 min bei Raumtemperatur. </li><li> Pellet in 1 ml Erythrozyten Lysepuffers und Inkubation auf Eis für 5 min auflysieren adhärenten PRBC, die nicht vom Gehirn konnte durch intrakardiale Perfusion entfernt werden. </li><li> 9 ml RPMI-Medium, waschen Zellen und Zentrifuge bei 250 × g für 10 min bei 4 ° C. </li><li> Resuspendieren BSL-enthaltende Pellet in 50-100 ul RPMI-Medium und Transfer Zellen zu einem Eppendorf-Röhrchen. </li><li> Verdünnte eine 5-10 ul Aliquots der Zellprobe 1:2 in Trypanblau zur Identifizierung von lebensfähigen Zellen. </li><li> Graf lebensfähigen Zellen unter dem Mikroskop mit einer Zählkammer. Von Tag 6 pi an, als P. berghei ANKA-infizierten Mäuse zeigen neurologische Symptome, 20.000-100.000 BSLs wiederhergestellt werden können. </li></ol> 4. Immunphänotypisierung der BSL durch Multicolour Durchflusszytometrie <ol><li> Zentrifuge Zellen bei 250 xg für 10 min bei 4 ° C, den Überstand aspirieren und Pellet in 50 ul Färbepuffer (PBS, 1% fötalem Kälberserum (FCS), 2 mm EDTA). </li><li> Fügen Sie die 1 ul gereinigt anti-CD16/32 Antikörper (Fcblock). </li><li> Inkubieren Zellen für 10 min auf Eis. </li><li> Waschen der Zellen mit 1 ml der Färbung Buffer. Zentrifuge Zellen bei 250 g für 10 min bei 4 ° C und saugt den Überstand. </li><li> Die Zellen in 50 ul Färbepuffer mit vorgegebenen optimalen Verdünnungen der erforderlichen fluoreszierenden Antikörpern, dh anti-CD4, anti-CD8, anti-TCR-und Anti-NK1.1. </li><li> Inkubieren für 1 Stunde auf Eis. </li><li> Waschen der Zellen mit 1 ml der Färbung Buffer. Zentrifuge Zellen bei 250 g für 10 min bei 4 ° C und saugt den Überstand. </li><li> Die Zellen in 100 ul PBS. </li><li> Übertragen Zellen einem Durchflusszytometrie Kunststoffrohr. </li><li> Mit einem Durchflusszytometer, erwerben mindestens 5.000-10.000 Ereignisse. Entsprechende ungefärbten Zelle steuert und Fluorochrom Entschädigung Proben enthalten, wie benötigt werden. </li><li> Analyse der Ergebnisse mit Hilfe geeigneter Durchflusszytometrie Software. </li></ol>

<ol>
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Die Isolierung und Analyse der BSL ist eine Methode, Charakterisierung und Quantifizierung von Entzündungszellen die Migration auf das Gehirn als Reaktion auf Gewebeverletzung oder Infektion in experimentellen Mausmodellen können. Die Einführung eines intrakardialen Perfusion Schritt zur Entfernung des Organs Blutabteil vor Extraktion und anschließende Zellisolation ist nützlich, um eine Kontamination von entzündlichen Zellen mit nicht-entzündliche zirkulierenden Leukozyten zu verhindern. Dies könnte nicht eine ...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare (optional) </td></tr><tr><td></td><td></td><td></td><td> Lösungen und Puffer </td></tr><tr><td> Giemsas azur Eosin-Methylenblau-Lösung </td><td> Merck Millipore </td><td> 1.09204.0500 </td><td> 1:10-Verdünnung in destilliertem Wasser </td></tr><tr><td> RPMI-Medium </td><td></td><td></td><td> Maus Tonus </td></tr><tr><td> Maus Tonus PBS </td><td></td><td></td><td> 20 mM Natriumphosphat, 0,149 NaCl, pH 7,3 </td></tr><tr><td> 0,4% Trypanblau </td><td> Sigma Aldrich </td><td> T-8154 </td><td> 1:2 Verdünnung </td></tr><tr><td> Collagenase D </td><td> Worthington Biochemical </td><td></td><td></td></tr><tr><td> DeoxyribonuClease (DNAse) I </td><td> Sigma Aldrich </td><td> D4263-5VL </td><td> Von Rinderpankreas </td></tr><tr><td> Percoll </td><td> GE Healthcare </td><td> 17-0891-01 </td><td> 30% ige Lösung in PBS </td></tr><tr><td> Ultrapure Tris </td><td> Invitrogen </td><td> 15505-020 </td><td></td></tr><tr><td> Ammoniumchlorid (NH 4 Cl) </td><td> AnalaR </td><td> 10017 </td><td></td></tr><tr><td> Red Cell Lysis Buffer </td><td></td><td></td><td> 17 mM Tris, 14Nm NH 4 Cl, pH 7,2 </td></tr><tr><td> FCS </td><td> Gibco </td><td> 1009 </td><td></td></tr><tr><td> EDTA Dinatriumsalz </td><td> Merck </td><td> 10093.5V </td><td> 0,1 M, pH 7,2 </td></tr><tr><td></td><td></td><td></td><td> Antikörper und Konjugate </td></tr><tr><td> Anti-Maus-CD16/CD32 (Fc Block), Klon 2.4G2 </td><td> BD Pharmingen </td> &lt;td&gt; 553142 </td><td> 1 ul in 50 ul Färbepuffer (0.5mg/50 ml) </td></tr><tr><td> FITC-anti-Maus-CD4, Klon H129.19 </td><td> BD Pharmingen </td><td> 553651 </td><td></td></tr><tr><td> PE-anti-Maus-NK1.1, Klon PK136 </td><td> BD Pharmingen </td><td> 553165 </td><td></td></tr><tr><td> PerCPCy5.5-Anti-Maus-CD8, Klon 53-6-7 </td><td> BD Pharmingen </td><td> 551162 </td><td></td></tr><tr><td> APC-anti-Maus-TCR-β, Klon H57-597 </td><td> BD Pharmingen </td><td> 553174 </td><td></td></tr><tr><td> PE-anti-Maus-CXCR3, Klon 220.803 </td><td> R &amp; D Systems </td><td> FAB1685P </td><td></td></tr><tr><td> Biotinyliertem Anti-Maus-CCR5, Klon C34-3448 </td><td> BD Pharmingen </td><td> 559922 </td><td></td></tr><tr><td> Streptavidin-PerCP-Cy5.5 </td><td> BD Pharmingen </td><td> 551419 </td><td></td></tr><tr><td></td><td></td><td></td><td> Ausrüstung und Material </td></tr><tr><td> SuperFrost Mikroskopobjektträger </td><td> Lomb Menzel-Gläser </td><td></td><td></td></tr><tr><td> Dissection Zangen, Scheren </td><td> REDA Instrumente </td><td></td><td></td></tr><tr><td> 500 ml PBS Reservoir </td><td> Nalgene </td><td></td><td></td></tr><tr><td> Gummischlauch </td><td></td><td></td><td></td></tr><tr><td> 23G Nadel </td><td> BD PrecisionGlide </td><td> 302008 </td><td></td></tr><tr><td> Zelldissoziationslösung Kit mit Metallsieb </td><td> Sigma Aldrich </td><td> CD-1 </td><td></td></tr><tr><td> 70 um Nylon Zellsieb </td><td> BD Falcon </td><td> 352350 </td><td></td></tr><tr><td> Hemocytometer </td><td> GmbH Neubauer </td><td> 717810 </td><td></td></tr><tr><td> Durchflusszytometrie </td><td> BD Falcon </td><td> 352008 </td><td></td></tr></tbody></table>

isolation and analysis of brain-sequestered leukocytes from plasmodium berghei anka-infected mice

Wir beschreiben ein Verfahren zur Isolierung und Charakterisierung von adhärenten Entzündungszellen aus Gehirn Blutgefäße von P. berghei ANKA-infizierten Mäusen. Infektion von empfänglichen Maus-Stämme mit diesen Parasiten Dehnung ergibt sich bei der Induktion von experimenteller zerebrale Malaria, eine neurologische Syndrom daß rekapituliert bestimmte wichtige Aspekte des Plasmodium falciparum-vermittelte schwerer Malaria beim Menschen 1,2. Reifen Formen von Blut-stufigen Malaria exprimieren parasitäre Proteine ​​auf der Oberfläche der infizierten Erythrozyten, wodurch sie an vaskulären Endothelzellen binden können. Dieser Prozess induziert Hindernisse in den Blutfluss, was zu Sauerstoffmangel und Blutungen 3 und stimuliert auch die Rekrutierung von Entzündungszellen Leukozyten an den Ort der Parasit Sequestrierung. Im Gegensatz zu anderen Infektionen, also Viren neutrotopic 4-6, beide Malaria-Parasiten befallenen Erythrozyten (PRBC) sowie zugehörigen InflammAtory Leukozyten bleiben abgesondert innerhalb von Blutgefäßen als Infiltration des Hirnparenchym. So eine Kontamination des einsamen Leukozyten mit nicht-entzündlichen zirkulierenden Zellen, umfangreiche intrakardiale Perfusion von infizierten-Mäuse vor Organentnahme und Gewebe Verarbeitung zu vermeiden wird in diesem Verfahren erforderlich, um die Blut-Kompartiment zu entfernen. Nach Perfusion, werden geerntet und seziert Gehirne in kleine Stücke. Das Tissue-Struktur wird weiter durch enzymatische Behandlung mit Kollagenase D und DNAse I gestört Die resultierende Hirnhomogenisat wird dann auf einem Percoll-Gradienten, die Trennung von brain-sequestered Leukozyten (BSL) von Myelin und andere Gewebedebris ermöglicht zentrifugiert. Isolierte Zellen werden dann gewaschen, gezählt unter Verwendung eines Hämozytometers und gefärbt mit fluoreszierenden Antikörpern für die anschließende Analyse durch Durchflusszytometrie. Dieses Verfahren ermöglicht eine umfassende phänotypische Charakterisierung von entzündlichen Leukozyten Migration auf das Gehirn in retion auf verschiedene Stimuli, einschließlich Schlaganfall sowie viralen oder parasitären Infektionen. Das Verfahren stellt auch ein nützliches Werkzeug für die Beurteilung von neuartigen anti-inflammatorische Therapien in präklinischen Tiermodellen.

Die Ergebnisse in Abb.. 2 zeigen Prozentsätze und absolute Zahl der verschiedenen BSL Populationen aus Gehirnen von perfundierten oder unperfused Malaria-infizierten und naiven Kontrollmäusen erholt. Isolated BSL wurden mit PE-anti-NK1.1 und APC-anti-TCR-β-Antikörper gefärbt, wie im Protokoll Text angezeigt. Übereinstimmend mit früheren Resultaten 7-9, bestehend αβTCR + T-Zellen einen hohen Anteil an der BSL Pool in Gehirnen von perfundierten Malaria-infizierte Mäuse (6 Tage pi). Diese Po...

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

Verfahren zur Isolierung von adhärenten entzündliche Leukozyten aus dem Gehirn Blutgefäße<em&gt; Plasmodium berghei</em&gt; ANKA-infizierten Mäusen beschrieben. Das Verfahren ermöglicht die Quantifizierung sowie phänotypischen Charakterisierung von isolierten Leukozyten nach Färbung mit fluoreszierenden Antikörpern und anschließende Analyse durch Durchflusszytometrie.

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. ...

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Immunology and Infection

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

12.7K Aufrufe.  The Walter and Eliza Hall Institute of Medical Research. Verfahren zur Isolierung von adhärenten entzündliche Leukozyten aus dem Gehirn Blutgefäße Plasmodium berghei ANKA-infizierten Mäusen beschrieben. Das Verfahren ermöglicht die Quantifizierung sowie phänotypischen Charakterisierung von isolierten Leukozyten nach Färbung mit fluoreszierenden Antikörpern und anschließende Analyse durch Durchflusszytometrie.

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

Isolation of Brain-infiltrating Leukocytes

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine encephalomyelitis virus (TMEV) via a rapid intracranial injection technique and how to purify a leukocyte-enriched population of infiltrating cells from whole brain. Briefly, mice are anesthetized with isoflurane in a closed chamber and are free-hand injected with a Hamilton syringe into the frontal cortex. Mice are then killed at various times after infection by isoflurane overdose and whole brains are extracted and homogenized in RPMI with a Tenbroeck tissue grinder. Brain homogenates are centrifuged through a continuous 30% Percoll gradient to remove the myelin and other cell debris. The cell suspension is then strained at 40 &mu;m, washed and centrifuged on a discontinuous Ficoll-Paque Plus gradient to select and purify the leukocytes. The leukocytes are then washed and resuspended in appropriate buffers for immunophenotyping by flow cytometry. Flow cytometry reveals a population of innate immune cells at the early stages of infection in C57BL/6 mice. At 24 hours post infection, multiple subsets of immune cells are present in the BILs, with an enriched population of Gr1+, CD11b+ and F4/80+cells. Therefore, this method is useful in characterizing the immune response to acute infection in the brain.

A rapid method to obtain infiltrating leukocytes from the murine brain is described. This method utilizes a continuous Percoll gradient and discontinuous Ficoll gradient to select and purify the leukocyte-enriched layer. Isolated leukocytes may then be characterized by flow cytometric measurements.

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 ...

Forschung

Immunologie und Infektion

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Eine experimentelle Modell zum Studium Tuberkulose-Koinfektion auf Natur Malaria Übertragung von Mycobacterium tuberculosis Und Plasmodium berghei</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

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 ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

Myeloischen Zellisolierung von Mäusehaut und ableitenden Lymphknoten Nach intradermale Impfung mit attenuierten Lebend<em&gt; Plasmodium</em&gt; Sporozoiten

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

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 und Analyse von Brain-sequestered Leukozyten aus