Les auteurs tiennent à remercier Mlle Liana Mackiewicz de l&#39;assistance technique. Ce travail a été rendu possible grâce au soutien du gouvernement l&#39;État de Victoria infrastructure opérationnelle et du gouvernement australien Santé nationale et du Conseil de recherches médicales IRIISS et de subvention de projet 1,031,212.

1. L&#39;infection des souris avec P. berghei ANKA- <ol><li> Décongeler une aliquote de cryoconservé P. berghei ANKA pRBC. </li><li> Retenir un paludisme cérébral résistant souris BALB / c donateurs (8-12 semaines d&#39;âge) en utilisant la technique de retenue à deux mains. Injecter la souris avec 100-200 ul de pRBC l&#39;aide d&#39;une seringue de 1 ml d&#39;insuline (28G aiguille). Régulièrement, 1-2 souris donneuses sont injectés. </li><li> Sur les 4-5 jours post-infection (pi) retirer souris donneuse de la cage et le placer sur une station de travail ou un tampon jetable de travail. </li><li> Doucement restreindre la souris par le bout de la queue et l&#39;utilisation de petits ciseaux couper le bout de la queue (environ 1 mm). Alternativement, une ponction petite queue à l&#39;aide d&#39;une aiguille 25 G pourrait être réalisée. </li><li> Déposer une goutte de sang de veine de la queue de la souris vers la fin d&#39;une lame de microscope dépoli fin. </li><li> Placez une deuxième lame (spreader) sur un angle de 45 ° et le sauvegarder dans la goutte de sang. Une fois le sang se propage le long du bord, pousser la veille épandeur-slideeul à travers la lame du microscope pour faire le frottis sanguin. Laisser le frottis sécher à l&#39;air. </li><li> Fixer les frottis sanguins dans le méthanol à 100% pendant 30 secondes, puis tache diapositives au Giemsa fraîchement préparée pendant 10 min. </li><li> Rincer frottis de sang avec l&#39;eau courante pendant 1 min, laisser sécher à l&#39;air et à examiner glissière sous le microscope (100x immersion dans l&#39;huile,). Énumérer pRBC à moins de 1000 érythrocytes et de calculer la parasitémie pour cent. Souris donneuses devraient atteindre des niveaux de parasitémie entre 2,5-5% avant d&#39;être saigné pour l&#39;infection des animaux de laboratoire. </li><li> Recueillir le sang de la souris donneuse par saignement rétro-orbitaire en utilisant un micro-hématocrite hépariné tube capillaire. Toutes les expériences sont réalisées en conformité avec les exigences Walter et Eliza Hall Institute du Comité d&#39;éthique animale. En fonction des besoins des animaux locaux comité d&#39;éthique procédures autres saignements peuvent être utilisés. Dissoudre la quantité requise de sang dans le milieu RPMI à une concentration finale de 1.10 6 pRBC/0.2 ml. Considérez queun hématocrite normal de la souris est de ~ 6x10 9 cellules rouges du sang / 1 ml. </li><li> Infect C57BL expérimentale / 6 souris (8-12 semaines) par injection intrapéritonéale (ip) 1x10 6 pRBC/0.2 ml. </li><li> Pour surveiller les niveaux de parasitémie de souris infectées suivez les étapes 1.3 à 1.8. Parasitémie doit être déterminé tous les 2-3 jours à partir du jour 2 ou 3 pi </li><li> L&#39;apparition du paludisme grave chez souris C57BL / 6 peut se manifester comme apparence voûtée, la gélinotte fourrure et une faible activité. Certaines souris peut recouvrer à ce stade, mais la progression de la perte de l&#39;auto-réflexe de redressement indique maladie irréversible et les animaux doivent être euthanasiés. </li></ol> 2. La perfusion intracardiaque de souris et d&#39;extraction du cerveau <ol><li> Assembler un système de perfusion par gravité manuel par fixation d&#39;un réservoir 500 ml d&#39;un support de colonne fixé à la partie supérieure de la colonne de support 60 cm. Raccorder des tubes en matière plastique à l&#39;extrémité inférieure du réservoir et assurer une pince de tube pour commander l&#39;écoulement tampon. Fixer le tube à une aiguille 23 G. Fill le réservoir avec du PBS (maintenue à 22 ° C), ouvrir et serrer permettre tampon afin de fonctionner à travers le tube pour éliminer toutes les bulles d&#39;air. </li><li> Euthanasier P. berghei ANKA-souris infectée par inhalation de CO 2. Confirmer la mort par l&#39;absence de pédale, et les réponses des voies respiratoires orbital. </li><li> Pin souris euthanasiées par les pattes de derrière et devant dorsalement sur une planche à dissection mousse de polystyrène contenues dans un bac en plastique. Selon le Comité des animaux sous anesthésie locale exigences d&#39;éthique pourrait être administré avant le début de pefusion intracardiaque. </li><li> Essuyez la face ventrale avec de l&#39;éthanol à 70%. </li><li> Utilisez de grands ciseaux et des pinces pour ouvrir la peau le long de la ligne médiane pour exposer la cavité thoracique. Pli cutané et la broche sur les côtés. </li><li> Tenez le sternum avec des pinces fines et couper le diaphragme et le long des deux côtés du sternum couper les côtes. Prenez soin de ne pas endommager les gros vaisseaux sanguins. </li><li> La broche de la cage thoracique du sternum lâchement à côté de la tête. </li><li> Tenez esprit ventriculesh pince fine et soigneusement inciser oreillette droite avec des ciseaux fins. </li><li> Insérez l&#39;aiguille 23G attaché au système de perfusion par gravité dans le ventricule gauche vers l&#39;aorte ascendante tandis que le PBS est en cours d&#39;exécution. Insérez seulement 0,5 cm de la pointe de l&#39;aiguille. </li><li> Perfuser la souris pendant 5 min ou jusqu&#39;à ce que l&#39;effluent est clair. </li><li> Détacher la souris et le poser sur son abdomen. Essuyez la tête avec de l&#39;éthanol à 70%. </li><li> Utilisation de grands ciseaux, faire une coupe juste au-dessus de la zone de la moelle épinière cervicale. </li><li> Utilisez des ciseaux fins pour faire une médiane caudale-rostrale coupe à partir de la base du crâne et la peau d&#39;écart pour exposer le crâne. </li><li> Tenant la tête, placez les lames de ciseaux petits dans chaque cavité orbitaire et faire une coupure entre les orbites. </li><li> Faites une coupe longitudinale le long de la suture sagittale et détachez soigneusement le crâne sur chaque hémisphère du cerveau vers l&#39;extérieur. </li><li> Avec une spatule, soulevez doucement le cerveau et le placer dans un tube à centrifuger de 10 ml contenant du milieu RPMI. </li></ol>3. Isolement de Brain-séquestrés leucocytes (BSL) <ol><li> Travailler dans une enceinte de sécurité, le lieu fraîchement récoltées cerveau au-dessus d&#39;un tamis cellulaire en acier inoxydable (40-60 mesh) dans une boîte de Pétri contenant 3-5 ml de milieu de culture RPMI tissus. </li><li> Coupez le tissu cérébral en petits morceaux. </li><li> Appuyez sur de petits morceaux de tissu cérébral à travers le filtre cellulaire à l&#39;aide d&#39;un piston de cristal. </li><li> Transfert homogénat de cerveau dans un tube de 10 ml et centrifuger à 250 xg pendant 10 min à 4 ° C. </li><li> Dissoudre le culot dans 3 ml de milieu RPMI, contenant 0,05% de collagénase D et DNAse I. 2U/ml </li><li> Tournez mélange pendant 30 min à température ambiante. Éliminer les débris cellulaires en poussant le mélange à travers un tamis de nylon 70 um cellule et / ou par incubation sur de la glace pendant 5 min. </li><li> Lay homogénat de cerveau sur un coussin de 7 ml de Percoll 30% et centrifuger à 400 xg (sans freins) pendant 20 min à température ambiante. </li><li> Remettre en suspension le culot dans 1 ml de tampon de lyse des globules rouges du sang et incuber sur de la glace pendant 5 min àlyser pRBC adhérent, qui n&#39;a pu être retirée du cerveau par perfusion intracardiaque. </li><li> Ajouter 9 ml de milieu RPMI, laver les cellules et centrifuger à 250 xg pendant 10 min à 4 ° C. </li><li> Remettre en suspension BSL contenant du pellet dans 50-100 pi de milieu RPMI et transférer les cellules dans un tube Eppendorf. </li><li> Diluer une aliquote de 5-10 pi de l&#39;échantillon de cellules 1:2 dans du bleu trypan pour l&#39;identification de cellules viables. </li><li> Compter les cellules viables sous le microscope à l&#39;aide d&#39;un hémocytomètre. A partir du jour de pi 6, lorsque P. berghei ANKA-souris infectées présentent des signes neurologiques, 20,000-100,000 BSLs peut être récupéré. </li></ol> 4. Immunophénotypage de BSL en cytométrie en flux Multicolore <ol><li> Centrifuger les cellules à 250 xg pendant 10 min à 4 ° C, aspirer le surnageant et remettre en suspension le culot dans 50 ul de tampon de coloration (PBS, 1% sérum de veau foetal (FCS), 2 mM EDTA). </li><li> Ajouter le pl 1 sur purifiée anti-CD16/32 anticorps (Fcblock). </li><li> Incuber les cellules pendant 10 min sur la glace. </li><li> Laver les cellules avec 1 ml de tampon de coloration. Centrifuger les cellules à 250 xg pendant 10 min à 4 ° C et aspirer le surnageant. </li><li> Resuspendre les cellules dans 50 ul de tampon de coloration contenant prédéterminés dilutions optimales de exigées anticorps fluorescents, c&#39;est à dire anti-CD4, anti-CD8, anti-TCR et anti-NK1.1. </li><li> Incuber pendant 1 heure sur la glace. </li><li> Laver les cellules avec 1 ml de tampon de coloration. Centrifuger les cellules à 250 xg pendant 10 min à 4 ° C et aspirer le surnageant. </li><li> Remettre en suspension les cellules dans 100 ul de PBS. </li><li> Transférer des cellules dans un tube de plastique de cytométrie de flux. </li><li> L&#39;aide d&#39;un cytomètre en flux, accumuler au moins 5.000-10.000 événements. Des contrôles de cellules non colorées et des échantillons de rémunération fluorochromes doivent être incluses au besoin. </li><li> Analyser les résultats en utilisant un logiciel approprié cytométrie en flux. </li></ol>

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Aucun conflit d&#39;intérêt déclaré.

L&#39;isolement et l&#39;analyse de BSL est une méthode qui permet la caractérisation et la quantification des cellules inflammatoires qui migrent vers le cerveau en réponse à une lésion tissulaire ou d&#39;une infection chez des souris modèles expérimentaux. L&#39;introduction d&#39;une étape de perfusion intracardiaque pour le retrait du compartiment sanguin avant l&#39;extraction d&#39;organes et de l&#39;isolement cellulaire ultérieur est utile pour prévenir la contamination de cellules inflammatoires avec...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires (optionnel) </td></tr><tr><td></td><td></td><td></td><td> Solutions et tampons </td></tr><tr><td> Giemsa azur éosine solution de bleu de méthylène </td><td> Merck Millipore </td><td> 1.09204.0500 </td><td> Dilution 1:10 dans de l&#39;eau distillée </td></tr><tr><td> Milieu RPMI </td><td></td><td></td><td> Souris tonicité </td></tr><tr><td> Souris tonicité PBS </td><td></td><td></td><td> Phosphate de sodium 20 mM, 0,149 NaCl, pH 7,3 </td></tr><tr><td> 0,4% bleu trypan </td><td> Sigma Aldrich </td><td> T-8154 </td><td> Dilution 1:2 </td></tr><tr><td> Collagénase D </td><td> Biochimique Worthington </td><td></td><td></td></tr><tr><td> DeoxyribonuClease (DNAse) Je </td><td> Sigma Aldrich </td><td> D4263-5VL </td><td> À partir de pancréas de bovin </td></tr><tr><td> Percoll </td><td> GE Healthcare </td><td> 17-0891-01 </td><td> Solution à 30% dans du PBS </td></tr><tr><td> Ultrapure Tris </td><td> Invitrogen </td><td> 15505-020 </td><td></td></tr><tr><td> Le chlorure d&#39;ammonium (NH 4 Cl) </td><td> AnalaR </td><td> 10017 </td><td></td></tr><tr><td> Rouge tampon de lyse cellulaire </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> Sel disodique d&#39;EDTA </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> Les anticorps et les conjugués d&#39; </td></tr><tr><td> Anti-souris CD16/CD32 (Fc Block), clone 2.4G2 </td><td> BD Pharmingen </td> &lt;td&gt; 553142 </td><td> 1 pl dans 50 ul de tampon de coloration (0.5mg/50 ml) </td></tr><tr><td> FITC anti-souris CD4, clone H129.19 </td><td> BD Pharmingen </td><td> 553651 </td><td></td></tr><tr><td> PE anti-souris NK1.1, clone PK136 </td><td> BD Pharmingen </td><td> 553165 </td><td></td></tr><tr><td> PerCPCy5.5 anti-souris CD8, clone 53-6-7 </td><td> BD Pharmingen </td><td> 551162 </td><td></td></tr><tr><td> APC anti-souris TCR-β, clone H57-597 </td><td> BD Pharmingen </td><td> 553174 </td><td></td></tr><tr><td> PE anti-souris CXCR3, clone 220803 </td><td> R &amp; D Systems </td><td> FAB1685P </td><td></td></tr><tr><td> Biotinylé anti-souris CCR5, clone C34-3448 </td><td> BD Pharmingen </td><td> 559922 </td><td></td></tr><tr><td> Streptavidine-PerCP-Cy5.5 </td><td> BD Pharmingen </td><td> 551419 </td><td></td></tr><tr><td></td><td></td><td></td><td> Équipement et matériel </td></tr><tr><td> Lame de microscope SuperFrost </td><td> Lomb Menzel-Gläser </td><td></td><td></td></tr><tr><td> Pinces à dissection, ciseaux </td><td> REDA Instrumente </td><td></td><td></td></tr><tr><td> 500 ml de PBS réservoir </td><td> Nalgene </td><td></td><td></td></tr><tr><td> Tube en caoutchouc </td><td></td><td></td><td></td></tr><tr><td> Aiguille 23G </td><td> BD PrecisionGlide </td><td> 302008 </td><td></td></tr><tr><td> Kit de dissociation cellulaire contenant un métal tamis </td><td> Sigma Aldrich </td><td> CD-1 </td><td></td></tr><tr><td> 70 um filtre cellulaire en nylon </td><td> BD Falcon </td><td> 352350 </td><td></td></tr><tr><td> Hémocytomètre </td><td> Neubauer GmbH </td><td> 717810 </td><td></td></tr><tr><td> Tubes de cytométrie en flux </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

Nous décrivons une méthode pour l&#39;isolement et la caractérisation des cellules adhérentes inflammatoires des vaisseaux sanguins du cerveau de P. berghei ANKA-souris infectées. L&#39;infection des souris sensibles avec les souches cela se traduit souche de parasite dans l&#39;induction de la malaria cérébrale expérimentale, un syndrome neurologique qui récapitule certains aspects importants de Plasmodium falciparum à médiation paludisme grave chez les humains 1,2. Formes matures du sang étapes paludisme exprimer des protéines parasites sur la surface de l&#39;érythrocyte infecté, ce qui leur permet de se lier à des cellules endothéliales vasculaires. Ce processus induit obstruction du flux sanguin, ce qui entraîne une hypoxie et des hémorragies 3 et stimule également le recrutement des leucocytes inflammatoires au site de la séquestration du parasite. Contrairement à d&#39;autres infections, à savoir les virus neutrotopic 4-6, les deux paludisme globules rouges parasités (pRBC) ainsi que associé Inflammleucocytes Atory reste séquestré dans les vaisseaux sanguins plutôt que de s&#39;infiltrer dans le parenchyme cérébral. Ainsi, pour éviter la contamination des leucocytes séquestrés avec des non-inflammatoires des cellules circulantes, vaste perfusion intracardiaque de souris infectées par-avant prélèvement d&#39;organes et de traitement des tissus est nécessaire dans cette procédure pour retirer le compartiment sanguin. Après la perfusion, les cerveaux sont prélevés et disséqués en petits morceaux. La structure du tissu est encore perturbée par un traitement enzymatique avec de la collagénase D et DNAse I. L&#39;homogénat de cerveau résultant est ensuite centrifugé sur un gradient Percoll qui permet de séparer le cerveau séquestrés leucocytes (BSL) de la myéline et des débris d&#39;autres tissus. Les cellules isolées sont ensuite lavées, comptées à l&#39;aide d&#39;un hémocytomètre et colorées avec des anticorps fluorescents pour une analyse ultérieure par cytométrie en flux. Cette procédure permet la caractérisation phénotypique complète des leucocytes inflammatoires de migrer vers le cerveau reréponse à divers stimuli, y compris les accidents vasculaires cérébraux ainsi que les infections virales ou parasitaires. La méthode fournit également un outil utile pour l&#39;évaluation de nouveaux traitements anti-inflammatoires dans des modèles animaux précliniques.

Les résultats de la Fig. 2 pourcentages montrent et le nombre absolu des différentes populations BSL récupérés à partir de cerveaux de souris perfusés ou unperfused contrôle infectées par la malaria et naïf. Isolé BSL ont été colorés avec du PE-anti-NK1.1 et APC-anti-TCR-β anticorps comme indiqué dans le texte du Protocole. Conformément aux conclusions précédentes 7-9, αβTCR cellules T comprend une forte proportion de la piscine BSL dans le cerveau des perfusés infectés par l...

Watch this Scientific Journal Video about Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice at JoVE.com

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

Une méthode pour l&#39;isolement des adhérents leucocytes inflammatoires des vaisseaux sanguins du cerveau de<em&gt; Plasmodium berghei</em&gt; ANKA-souris infectées est décrite. La méthode permet la quantification ainsi que la caractérisation phénotypique des leucocytes isolés après coloration avec des anticorps fluorescents et leur analyse ultérieure par cytométrie en flux.

Isolement et analyse de Brain-séquestrés de leucocytes<em&gt; Plasmodium berghei</em&gt; ANKA-souris infectées

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 vues.  The Walter and Eliza Hall Institute of Medical Research. Une méthode pour l'isolement des adhérents leucocytes inflammatoires des vaisseaux sanguins du cerveau de Plasmodium berghei ANKA-souris infectées est décrite. La méthode permet la quantification ainsi que la caractérisation phénotypique des leucocytes isolés après coloration avec des anticorps fluorescents et leur analyse ultérieure par cytométrie en flux.

Vidéo: 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.

Isolement du cerveau infiltrant Leucocytes

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

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Immunologie et infection

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.

Un modèle expérimental pour étudier la tuberculose-paludisme co-infection à transmission naturelle de Mycobacterium tuberculosis Et 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.

Bioénergétique et les oxydatif: Protocoles pour l&#39;isolement et l&#39;évaluation des leucocytes humains et plaquettes

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.

Analyser les effets des cellules stromales sur le recrutement des leucocytes de débit

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.

Isolement des leucocytes à partir de l&#39;interface humaine materno-fœtale

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.

Isolement de leucocytes des tissus murins à l&#39;interface materno-foetale

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.

Isolement de leucocytes infiltrant de la peau de souris Utilisation de digestion enzymatique et séparation par gradient

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

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.

Isolement et analyse par cytométrie en flux des cellules immunitaires du cerveau ischémique souris

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

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.

Myéloïde cellule d&#39;isolement de la peau de souris et Vidange ganglion Après intradermique immunisation avec Live Atténuée<em&gt; Plasmodium</em&gt; sporozoïtes

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-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.

Récolte sélective de Marginating-hépatiques Leucocytes

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