Ce travail a été soutenu par la subvention NS64571 du NINDS (CLH), par une sentence de développement en début de carrière de la Mayo Clinic (CLH), et par un généreux don de Donald et Frances Herdrich (CLH). Nous tenons à remercier la Clinique Mayo de débit de base de cytométrie de l&#39;aide.

1. Injection du virus intracrânienne: La technique suivante a été modifié et largement utilisée par notre laboratoire et ses collègues. Brièvement, l&#39;injection intracrânienne de souche du Daniel de virus murin de Theiler encéphalomyélite (TMEV) ou fictive-infection (1, 10) est réalisée sur des souris jeunes (de préférence 5-6 semaines d&#39;âge) pour obtenir des leucocytes infiltrant le cerveau (Bils). S&#39;il vous plaît noter que les résultats diffèrent entre les souches du Daniel, la souche de soja et la souche GDVII. Aux fins de la récolte de Bils, souris reçoivent 2x10 5 UFP de TMEV et sont infectées pendant 24 heures. <ol><li> Fixez l&#39;aiguille d&#39;injection (27 gauge, ¼, Kendall), à une seringue automatique 1 ml Hamilton et réglée pour délivrer 10 ul de virus. </li><li> Dresser TMEV de 10 uL de DMEM dans la seringue avec une attention particulière aux bulles d&#39;air. Le cas échéant, des souris infectées reçoivent simulacre 10 ul de virus sans DMEM. </li><li> Juste avant l&#39;injection, verser environ 1 mL d&#39;isoflurane dans une cloche de verre qui contient une grille de fils de coton avec le substrat en dessous. </li><li> Placez la souris sur la grille de fil direclty dans la cloche jusqu&#39;à ce qu&#39;ils deviennent légèrement anesthésiés, insensibles aux pieds-pincement et immobilisé par l&#39;anesthésique par inhalation, environ 10-15 secondes. Souris ne devrait pas venir en contact direct avec l&#39;isoflurane, comme substance peut être irritante et caustique. </li><li> Alors que sous anesthésie, de préparer des souris pour injection par localiser rapidement des coordonnées appropriées dans la région du cortex frontal sur le crâne à l&#39;aide de rasage et de l&#39;encrage. L&#39;emplacement général pour l&#39;injection est de 1 mm en avant de bregma et 1 mm latéralement à la suture sagittale sur le côté droit (figure 1). Alors que l&#39;injection stéréotaxique peut être approprié pour certaines situations expérimentales, nous avons constaté que l&#39;injection à main levée, sans le rasage ou l&#39;encrage est approprié pour la majorité des expériences. Écarté les poils avec la pointe de l&#39;aiguille le long des deux axes est approprié pour l&#39;injection. </li><li> Pour faire une injection, d&#39;orienter l&#39;aiguille perpendiculaire au crâne et appuyez à travers l&#39;os d&#39;une profondeur d&#39;environ 3 mm. </li><li> Le virus Express dans le cerveau en appuyant sur le bouton seringue Hamilton vers le bas et attendre 2 secondes pour que le virus d&#39;entrer dans le cerveau avant de se retirer. </li><li> Retirer délicatement l&#39;aiguille et placer la souris dans un endroit propre, sécher la cage. Souris réveiller après une anesthésie plus rapidement et de reprendre un fonctionnement normal en quelques minutes. Accorder une attention adéquate à des souris qui présentent des symptômes anormaux, tels que des saignements excessifs du site d&#39;injection, comme l&#39;euthanasie de l&#39;animal peut être appropriée. Une bonne manipulation des animaux 6 et l&#39;adhésion aux National Institutes of Health et la protection des animaux et des directives institutionnelles Comité utilisation doit être suivie. </li></ol> 2. Brain-infiltrer la préparation des leucocytes: <ol><li> Préparer des tubes à fond rond Oak Ridge centrifugeuse qui peut accueillir une capacité de 30 ml avec 10 ml de RPMI 1640, 9 ml de Percoll et 1 ml de PBS 10X et les tubes doivent s&#39;asseoir à la RT sur le banc pendant le processus d&#39;élimination du cerveau. </li><li> Apportez Ficoll-Paque Plus à température ambiante (TA). </li><li> Euthanasier des animaux par l&#39;isoflurane modifiés surdosage 5 et supprimer rapidement le cerveau à l&#39;aide d&#39;une spatule en acier inoxydable de tissu et le placer dans un tube de 15 ml conique contenant 5 ml de RPMI sur la glace. Dans certaines circonstances, PBS-perfusion peut être approprié de retirer des cellules circulantes du sang périphérique de la préparation de Bils. Les souris doivent être pleinement et correctement anesthésié avant PBS-perfusion. </li><li> Transfert du cerveau et de la solution RPMI à un verre de 7 ml meuleuse Pyrex marque tissus Tenbroeck et doucement homogénéiser avec 10-15 coups. </li><li> Pipet un supplément de 5 ml de RPMI dans l&#39;homogénéisateur et une pipette de haut en bas deux fois. Transfert du cerveau homogénat (env. 10 ml) d&#39;préalablement préparé à fond rond tube et retourner doucement 2-3 fois pour mélanger. </li><li> Spin homogénat de cerveau à 7800g Ave pendant 30 min à température ambiante dans une F0360 rotor angulaire dans un Beckman Allegra X-22R centrifugeuse clinique. </li><li> Après centrifugation, enlever les débris de myéline qui a flotté au sommet de la pente. Recueillir la couche de leucocytes qui flotte au-dessus des globules rouges culot (figure 2). </li><li> Strain couche de leucocytes à travers un tamis 40 um cellule dans une forme conique de 50 ml. Apporter une solution à un volume de 50 ml avec IPMB. </li><li> Tubes contenant Spin suspension cellulaire diluée dans une centrifugeuse clinique à 1500 rpm (600g AVE) pendant 5 min à température ambiante. </li><li> Aspirer le surnageant et recueillir le culot cellulaire. Reprendre le culot dans 1 ml de tampon FACS (1% de sérum albumine bovine, azoture de sodium à 0,02% en calcium et en magnésium sans PBS) et le transfert de la suspension cellulaire à 5 ml tubes ronds FACS bas. </li><li> Suspension de sous-couche soigneusement avec 1 ml de Ficoll-Paque Plus. </li><li> Spin tube à 2500 rpm (1400 a donné) en centrifugeuse clinique pendant 25 min à température ambiante sans frein. </li><li> Retirer couche blanche duveteuse à l&#39;interface (figure 2) avec une pipette ml et transférer à nouveau tube de 5 ml FACS. Lavez caunes avec 4 ml de tampon FACS. </li><li> Spin tubes à 1500 rpm (600g AVE) pendant 5 min à température ambiante dans centrifugeuse clinique pour sédimenter les cellules. </li><li> Aspirer le surnageant et recueillir culot cellulaire. Reprendre le culot dans leucocytes tampon approprié et garder sur la glace dans FACS nouveau tube. </li><li> Compter les cellules, d&#39;évaluer la viabilité cellulaire par exclusion du bleu Trypan et analyser par cytométrie en flux. En général, les enquêteurs peuvent s&#39;attendre à acquérir environ 5 x 10 5 cellules / animal. </li></ol> 3. Immunophénotypage par cytométrie en flux <ol><li> Après avoir isolé et le comptage des leucocytes, le spin des cellules pendant 3 minutes à 1500 rpm (600 a) dans une centrifugeuse clinique. </li><li> Reprendre le culot dans un tampon bloquant contenant les éléments suivants: tampon FACS, surnageant de 2.4G2 hybridomes (Fc bloc; anti-CD16/32) et sérum de veau fœtal à un ratio de 10:05:01 et incuber pendant 30 minutes à 4 ° C . </li><li> Préparer et ajouter des anticorps conjugués dirigés contre les antigènes extracellulaires aux cellules bloquées à une concentration de 1:200 et incuber pendant 30 minutes à 4 ° C dans l&#39;obscurité. Stain et incuber les cellules dans 200 ul dans un 96 puits à fond en V assiette. Des contrôles appropriés incluent cellules non colorées et des échantillons de compensation fluorochrome, le cas échéant. </li><li> Faites tourner les cellules colorées à la température ambiante pendant 3 minutes à 1500 rpm (600 a) dans une centrifugeuse clinique appropriée en utilisant un rotor pour plaques de 96 puits. </li><li> Aspirer le surnageant et laver les cellules avec 200 ul de tampon FACS par un léger pipetage haut et en bas trois fois. </li><li> Répétez la technique de lavage ci-dessus à deux reprises. </li><li> Après le lavage, remettre en suspension les cellules dans du paraformaldéhyde à 2% et le transfert à des tubes FACS. La fixation est nécessaire pour l&#39;analyse par cytométrie en flux de cellules infectées de niveau de biosécurité 2 réactifs. </li><li> Exécuter les cellules fixées sur une BD FACS Calibur suivant une méthode de cytométrie en flux 8 modifiés et d&#39;analyser les fichiers hors connexion en utilisant FlowJo, WinMDI ou d&#39;autres logiciels disponibles analyse de cytométrie en flux. </li><li> L&#39;analyse des événements par 50-100,000 échantillon est généralement requis pour l&#39;immunophénotypage adéquate. </li></ol> Anticorps directement conjugués utilisés dans cette expérience: CD45 a été détecté avec clone 30-F11. Ly6C / G a été détecté avec clone Gr1, RB6-8C5. CD11b a été détecté avec clone M1/70. F4/80 a été détecté avec clone BM8. 4. Les résultats représentatifs: Nous montrons les résultats de phénotypage de cellules pour Bils souris à 24 heures après l&#39;infection (figure 3). Les leucocytes ont été colorés avec PerCP conjugué anti-souris CD45 pour détecter les cellules immunitaires, PE-anti-souris conjugué Ly6C / G pour détecter les monocytes inflammatoires, APC-anti-souris conjugué CD11b et APC-conjugué anti-souris pour détecter les cellules F4/80 de la lignée monocytaire. L&#39;analyse a été réalisée avec un instrument BD FACS Calibur. <img src="/files/ftp_upload/2747/2747fig1.jpg" alt="Figure 1"/> Figure 1. Localisation anatomique du site d&#39;injection. Le site d&#39;injection se situe à 1 mm en avant de bregma et 1 mm latéralement à la suture sagittale sur le côté droit. <img src="/files/ftp_upload/2747/2747fig2.jpg" alt="Figure 2"/> Figure 2. Leucocytes Illustration de séparation par gradient de leucocytes et de Bils. Sont initialement collectées directement en dessous de la couche de débris de myéline et surtout le culot de RBC dans le gradient de Percoll. La couche blanche duveteuse (Bils) à l&#39;interface est recueillie à partir du gradient de Ficoll. <img src="/files/ftp_upload/2747/2747fig3.jpg" alt="Figure 3"/> Figure 3. Immunophénotype des cerveaux infilatrating leucocytes à 24 heures post-infection leucocytes souris. Ont été isolés à partir du cerveau par centrifugation densité différentielle du tissu homogénats préparés à partir d&#39;animaux 24 h après l&#39;infection intracrânienne. Les cellules ont été colorées avec des anticorps par fluorescence-conjugués contre les souris CD45, Ly6C / G, CD11b, et F4/80. Gating initiale a été réalisée en traçant CD45, un marqueur de cellules immunitaires, contre prodiffusion (FSC), un indicateur de la taille des cellules (C). Le Salut CD45 (A, D), CD45 lo (B, E), et CD45 neg (F, G), les populations ont ensuite été analysés pour les niveaux d&#39;expression relative des marqueurs monocytes et les macrophages Ly6C / G, F4/80 (A, B , F), et CD11b (D, E, G). Ly6C / G + + CD11b + F4/80 monocytes inflammatoires ont été trouvés presque exclusivement dans la population CD45 Salut (A, D), compatible avec l&#39;infiltration de ces cellules de la périphérie. En revanche, Ly6C/G-F4/80 lo CD11b + macrophages ont été trouvés presque exclusivement dans le lo CD45 (B, E) et CD45 neg (G) des populations, compatible avec un phénotype résident. Dans de nombreuses expériences, nous n&#39;avons jamais observé de cellules CD45 salut ou Ly6C / G + + CD11b + F4/80 monocytes inflammatoires dans le cerveau des infectés ou fictive-infectionTed souris (données non présentées).

<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>

Nous utilisons régulièrement la cytométrie en flux pour déterminer la qualité de la préparation des cellules du cerveau infiltrer, et de distinguer les différentes populations de cellules immunitaires 2, 9. A aiguë du temps des points, notre méthode Bils rendements des pourcentages élevés de monocytes inflammatoires au sein de la population CD45hi ainsi que de forts pourcentages de macrophages dans la population CD45lo. Cela indique que la réponse immunitaire dans reproductibles le cerveau peut ê...

<table border="1"><tbody><tr><th> Réactifs </th><th> Société </th><th> Le numéro de catalogue </th><th> Commentaires </th></tr><tr><td> TMEV </td><td> Howe Lab </td><td> N / A </td><td></td></tr><tr><td> Daniel souche </td><td> Invitrogen </td><td> 21063-029 </td><td></td></tr><tr><td> isoflurane </td><td> Novaplus </td><td> NDC 0409-3292-49 </td><td></td></tr><tr><td> à fond rond tubes Oak Ridge centrifugeuse </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> PBS 10X </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> Bleu Trypan 0,4% (p / v) </td><td> Mediatech </td><td> 25 à 900-CI </td><td></td></tr><tr><td> 15 ml tubes coniques </td><td> BD Falcon </td><td> 352097 </td><td></td></tr><tr><td> 7 ml en verre pyrex marque broyeur de tissus Tenbroeck </td><td> Fisher </td><td> 08 à 414-10B </td><td></td></tr><tr><td> 40 tamis cellulaire um </td><td> BD Falcon </td><td> 352340 </td><td></td></tr><tr><td> Conique de 50 ml </td><td> BD Falcon </td><td> 352070 </td><td></td></tr><tr><td> l&#39;albumine sérique bovine </td><td> Sigma </td><td> A9647 </td><td></td></tr><tr><td> azide de sodium </td><td> Sigma </td><td> S8032 </td><td></td></tr><tr><td> CMF-PBS </td><td> Mediatech </td><td> 21 à 040-CV </td><td></td></tr><tr><td> FACS tubes </td><td> BD Falcon </td><td> 352054 </td><td></td></tr><tr><td> sérum de veau fœtal </td><td> Sigma </td><td> F4135 </td><td></td></tr><tr><td> 2.4G2 hybridomes </td><td> ATCC </td><td> HB-197 </td><td></td></tr><tr><td> Costar à fond en V plateau </td><td> Corning </td><td> 3894 </td><td></td></tr><tr><td> Allegra X-22R centrifugeuse ou équivalent </td><td> Beckman </td><td> N / A </td><td></td></tr><tr><td> 96 puits, seau à la plaque et rotor </td><td> Beckman </td><td> S2096 </td><td></td></tr><tr><td> Rotor angulaire </td><td> Beckman </td><td> F0360 </td><td></td></tr><tr><td> paraformaldéhyde </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> Arbre Star, Inc </td><td> N / A </td><td></td></tr><tr><td> CD45 </td><td> BD Biosciences </td><td> 557235 </td><td> clone: ​​30-F11 </td></tr><tr><td> Gr1 (Ly6C / G) </td><td> BD Biosciences </td><td> 553128 </td><td> clone: ​​RB6-8C5 </td></tr><tr><td> CD11b </td><td> ebiosciences </td><td> 17-0112-83 </td><td> clone: ​​M1/70 </td></tr><tr><td> F4/80 </td><td> ebiosciences </td><td> 17-4801-82 </td><td> clone: ​​BM8 </td></tr></tbody></table>

isolation of brain-infiltrating leukocytes

Nous décrivons une méthode pour la préparation de leucocytes infiltrant le cerveau (Bils) de souris. Nous démontrons comment infecter des souris avec le virus murin de Theiler encéphalomyélite (TMEV) via une technique d&#39;injection intracrânienne rapide et comment purifier une population leucocytaire enrichi des cellules infiltrant du cerveau entier. Brièvement, les souris sont anesthésiés à l&#39;isoflurane dans une chambre fermée et sont main-libre injecté avec une seringue Hamilton dans le cortex frontal. Les souris sont ensuite tués à différents moments après l&#39;infection par surdose isoflurane et cerveaux entiers sont extraits et homogénéisé dans du RPMI avec un broyeur de tissus Tenbroeck. Homogénats de cerveaux sont centrifugés à travers un gradient de Percoll à 30% continue d&#39;éliminer les débris cellulaires et d&#39;autres myéline. La suspension cellulaire est alors tendu à 40 um, lavés et centrifugés sur un discontinu de Ficoll-Paque plus dégradé à sélectionner et à purifier les leucocytes. Les leucocytes sont ensuite lavées et resuspendues dans des tampons appropriés pour l&#39;immunophénotypage par cytométrie en flux. La cytométrie en flux révèle une population de cellules immunitaires innées aux stades précoces de l&#39;infection chez les souris C57BL / 6. A 24 heures après l&#39;infection, multiples sous-ensembles de cellules immunitaires sont présentes dans le Bils, avec une population enrichie de Gr1 +, CD11b + et + F4/80 cellules. Par conséquent, cette méthode est utile pour caractériser la réponse immunitaire à une infection aiguë dans le cerveau.

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

Isolation of Brain-infiltrating Leukocytes

Une méthode rapide pour obtenir leucocytes infiltrants du cerveau murin est décrite. Cette méthode utilise un gradient de Percoll continue et gradient de Ficoll discontinu pour sélectionner et purifier la couche leuco-enrichi. Leucocytes isolés peuvent alors être caractérisées par des mesures de cytométrie en flux.

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 ...

isolation-of-brain-infiltrating-leukocytes

Research

JoVE Journal

Immunology and Infection

19.0K vues.  Mayo Clinic College of Medicine. Une méthode rapide pour obtenir leucocytes infiltrants du cerveau murin est décrite. Cette méthode utilise un gradient de Percoll continue et gradient de Ficoll discontinu pour sélectionner et purifier la couche leuco-enrichi. Leucocytes isolés peuvent alors être caractérisées par des mesures de cytométrie en flux.

Vidéo: 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.

Isolement des cellules immunitaires fonctionnelles cardiaques

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 ...

Recherche

Immunologie et infection

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.

L&#39;isolement des cellules de souris poumon dendritiques

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.

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. ...

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 ...

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.

Récolte sélective de Marginating-pulmonaires Leucocytes

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.

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 ...