Ce travail a été financé par une subvention de recherche et une subvention en aide à partir du projet nanotechnologie alimentaire du Ministère de l&#39;Agriculture, des Forêts et des Pêches du Japon.

1. Perfusion du foie <ol><li> Anesthetize un adulte mâle Sprague-Dawley rats (de 10 à 15 semaines, âgé; poids corporel 150-200 g) par une injection intrapéritonéale d&#39;une solution de pentobarbital de sodium (poids 50 mg de sodium pentobarbital / kg). </li><li> Placez l&#39;animal dans une casserole inoxydable et de pulvérisation d&#39;éthanol à 70% sur son abdomen et du thorax. </li><li> Faire une petite entaille et retirez la peau. Ouvrez l&#39;abdomen par le couper avec une paire de ciseaux. </li><li> Confirmez l&#39;emplacement de la veine porte, passer un fil chirurgical dans la veine porte et lâche bouquet. </li><li> Clump la partie distale de la veine cave inférieure et la veine mésentérique pour empêcher le reflux sanguin dans le foie. Ouvrez la cage thoracique, en coupant la membrane avec des ciseaux, et exposer le cœur. </li><li> Insérer un cathéter en plastique (2 mm de diamètre) dans la veine porte et serrez fermement le fil autour du cathéter. </li><li> Connecter le cathéter à un système de perfusion qui est chargé avec une solution de lavage (Ca 2 +-Mg 2 + HBSS libre contenant 0,5 mM, EGTA 10 mM d&#39;HEPES et 4,2 mM NaHCO 3; pH 7,2) préchauffée à 37 ° C. </li><li> Commencez la perfusion in situ à un taux de 10 ml / min. Dans le même temps, faire une coupe dans le mur de l&#39;oreillette droite. Poursuivre la perfusion pendant 10 min. </li><li> Mettez la solution de perfusion à la solution de collagénase [HBSS contenant HEPES 10 mM et de 4,2 mm de NaHCO 3 complété par la collagénase de type I (0,05%) et inhibiteur de la trypsine (50 pg / ml); pH 7,5], et perfuser pendant 10 - 20 min à un taux de 10 ml / min jusqu&#39;à ce que le foie gonfle légèrement et montre le signe de la désintégration de la structure du foie sous la membrane séreuse. </li></ol> 2. Préparation de la fraction de cellules du parenchyme hépatocytaire riches <ol><li> Retirer et transférer soigneusement le foie dans un bécher stérile contenant 25 ml de solution de collagénase. Hacher en petits morceaux avec des ciseaux. </li><li> Ajouter 75 ml de MEM froid dans la suspension cellulaire résultante. Après pipetage doux, filtrat à travers un tamis cellulaire (100 um) pour enlever les tissus conjonctifs et des fragments de tissus non digérés. </li><li> Transférer le filtrat dans 50 ml tubes coniques et centrifuger à 50 g pendant 1 min à 4 ° C (frein off). </li><li> Soigneusement jeter le surnageant. Reprendre le culot avec du MEM froid. </li><li> Répétez 2,4) à trois reprises. </li></ol> 3. Mixte culture primaire de cellules du foie <ol><li> Suspendre les cellules obtenues à 2,5) dans le milieu de croissance composé de DMEM contenant 10% de la chaleur FCS inactivé, supplémenté avec 100 uM β-mercaptoéthanol, 10 pg / ml d&#39;insuline, 100 ug / ml de streptomycine et 100 U / ml de pénicilline, et la semence en cinq à dix flacons de culture tissulaire (superficie: 75 cm 2) à une densité de 6,7 x 10 4 cellules / cm 2 (5 x 10 6 cellules / flacon / 12 -15 ml de milieu). </li><li> Incuber la culture flasksat 37 ° C dans une atmosphère de 5% CO 2 - 95% d&#39;air. </li><li> Remplacez le milieu de croissance aux 2 à 3 jours d&#39;intervalle. </li></ol> 4. Isolement sélectif des macrophages <ol><li> Après 12 jours de culture, les macrophages prolifèrent sur la feuille de cellule. Ces cellules sont suspendues par la réciprocité secouer les flacons de culture à 80 - 120 coups par minute pendant 30 min à 37 ° C. </li><li> Transférer le milieu de culture en 100 mm non de culture de tissus de qualité des plats en plastique. Piscine de la moyenne de deux flacons T75 dans un plat en plastique. Flacons de culture Recharge avec le milieu de croissance et les retourner à l&#39;incubateur. </li><li> Incuber les plats en plastique pendant 30 min à 37 ° C dans une atmosphère de 5% de CO 2 à 95% d&#39;air. Les macrophages facilement attacher à des tissus non des boîtes de culture plastique de qualité en vertu du présent processus d&#39;incubation, tandis que d&#39;autres cellules contaminantes pas. </li><li> Aspirer le milieu et rincer doucement le plat de plastique avec du PBS. Répétez cette étape trois fois. </li><li> Ajouter 1 ml de solution de TrypLE express dans le plat, et incuber pendant 10min à 37 ° C dans une atmosphère de 5% de CO 2 à 95% d&#39;air. </li><li> Ajouter 9 ml de milieu de croissance. Grattez délicatement les macrophages attachés par un racleur de cellules. </li><li> Transférer la suspension cellulaire dans un tube de 15 ml conique et centrifuger à 180 xg pendant 5 min à 25 ° C (frein). </li><li> Jeter le surnageant et ajouter 1 ml de milieu de croissance. Dissocient en cellules individuelles par pipetage vigoureux. Énumérer le nombre de cellules par un hémocytomètre comptage. </li><li> Étapes d&#39;isolement peut être répétée avec les mêmes flacons de culture de deux à trois jours d&#39;intervalle pendant plus de deux semaines. </li><li> Macrophages isolés peuvent être cultivées dans le milieu de croissance décrit dans 3.1. </li></ol> 5. Les résultats représentatifs Un exemple d&#39;une culture mixte primaire de cellules adultes de foie de rat est montré dans la figure 2. Hépatocytes parenchymateux perdent la morphologie des cellules épithéliales dans les quelques jours de culture, de dégénérer ou se transformer en fibroblastes comme des cellules.Puis, autour du jour 6 à 12, contraste de phase-lumineux, rond cellules de type macrophage vigourouly prolifèrent sur la feuille de la cellule fibroblastique. En secouant les flacons de culture, les macrophages sont facilement en suspension dans le milieu de culture, et le transfert subséquent et l&#39;incubation dans des plats en plastique dans le résultat d&#39;adhésion sélective des macrophages (figure 3). Les cellules de type macrophage peuvent être récoltées dès le jour 8, et les chiffres ont atteint des niveaux maximaux sur les jours 12 à 14. Plus de 10 6 cellules peuvent être récoltées à partir d&#39;un flacon T75 culture répétée 2 à 3 jours d&#39;intervalle pendant plus de deux semaines, permettant un rendement total de cellules par flacon de 10 7 par T75 flacon de culture (figure 3). Les puretés des macrophages isolés étaient de 95 à 99%, tel qu&#39;évalué par flowcytometry ou immunocytochimie avec des rats macrophages anticorps spécifiques, tels que ED-1 (figure 4), ED-3, OX-41 (figure 4) et Iba1 9. Les cellules isolées montrent des caractéristiques typiques des macrophages, comme la phagocytose active de perles polystylene (figure 5), réponse proliférative à la GM-CSF recombinant, la sécrétion des maladies inflammatoires / anti-inflammatoires des cytokines après stimulation par le LPS, et la formation de cellules géantes multinucléées 9. <img src="/files/ftp_upload/2757/2757fig1.jpg" alt="Figure 1" /> Figure 1. Un système d&#39;isolement sélectif et de purification des cellules de type macrophage de la culture de cellules primaires mixtes de foie de rat. <img src="/files/ftp_upload/2757/2757fig2.jpg" alt="Figure 2" /> Figure 2. Culture primaire de cellules de foie de rat et la prolifération des cellules de type macrophage. La flèche indique une cellule géante multinucléaire. Barre d&#39;échelle = 100 pm. Reproduit de la Revue des méthodes immunologiques, Vol.360, 47-55 (2010) 9 avec la permission d&#39;Elsevier. <img src="/files/ftp_upload/2757/2757fig3.jpg" alt="Figure 3" /> Figure 3. Isolement sélectif des cellules de type macrophage par le tremblement et la méthode de fixation. Les cellules ont été suspendues dans le milieu de culture en secouant les flacons, par la suite transférés dans des boîtes de culture de tissu non plastique de qualité, et incubées à 37 ° C. Dès 10 min après le placage, cellules de type macrophage attaché à la surface plat, tandis que d&#39;autres cellules fibroblastiques contaminant est resté suspendu. Après rinçage avec du PBS, une population de macrophages hautement purifiée a été obtenue. Ces cellules gagné la morphologie des macrophages typiques après 40 min de la culture, et les cellules mitotiques ont été fréquemment observés (flèches). Les variations ultérieures de nombre de cellules récupérées à partir des flacons à des périodes différentes cultures sont représentées. Les valeurs sont une moyenne ± écart de trois à cinq flacons. Des expériences ont été répétés trois fois et les données sont indiquées par des symboles différents. Barre d&#39;échelle = 100 pm. Reproduit de la Revue des méthodes immunologiques, Vol.360, 47-55 (2010) 9 avec la permission d&#39;Elsevier. <img src="/files/ftp_upload/2757/2757fig4.jpg" alt="Figure 4" /> Figure 4. Coloration immunocytochimique des cellules de type macrophage avec des anticorps monoclonaux contre les macrophages de rat. <img src="/files/ftp_upload/2757/2757fig5.jpg" alt="Figure 5" /> Figure 5. Phagocytose des microbilles marqué FITC par cellules de type macrophage.

<ol>
	<li>Crispe, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+liver+as+a+lymphoid+organ.">The liver as a lymphoid organ.</a> Annu. Rev. Immunol. 27, 147-163 (2009).</li><li>Roberts, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+Kupffer+cell+in+mediating+hepatic+toxicity+and+carcinogenesis.">Role of the Kupffer cell in mediating hepatic toxicity and carcinogenesis.</a> Toxicol. Sci. 96, 2-15 (2007).</li><li>Gao, B., Jeong, W. I., Tian, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver:+An+organ+with+predominant+innate+immunity.">Liver: An organ with predominant innate immunity.</a> Hepatology. 47, 729-736 (2008).</li><li>Janousek, J., Strmen, E., Gervais, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+murine+Kupffer+cells+by+centrifugal+elutriation.">Purification of murine Kupffer cells by centrifugal elutriation.</a> J. Immunol. Methods. 164, 109-117 (1993).</li><li>Olynyk, J. K., Clarke, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+primary+culture+of+rat+Kupffer+cells.">Isolation and primary culture of rat Kupffer cells.</a> J. Gastroenterol. Hepatol. 13, 842-845 (1998).</li><li>Heuff, G., Meyer, S., Beelen, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+rat+and+human+Kupffer+cells+by+a+modified+enzymatic+assay.">Isolation of rat and human Kupffer cells by a modified enzymatic assay.</a> J. Immunol. Methods. 174, 61-65 (1994).</li><li>Seglen, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+isolated+rat+liver+cells.">Preparation of isolated rat liver cells.</a> Methods Cell Biol. 13, 29-83 (1976).</li><li>Yoshioka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+and+expression+of+cytokine+mRNAs+by+lipopolysaccharide+in+bovine+Kupffer+cells.">Primary culture and expression of cytokine mRNAs by lipopolysaccharide in bovine Kupffer cells.</a> Vet. Immunol. Immunopathol. 58, 155-163 (1997).</li><li>Kitani, H., Takenouchi, T., Sato, M., Yoshioka, M., Yamanaka, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+isolation+method+for+macrophage-like+cells+from+mixed+primary+cultures+of+adult+rat+liver+cells.">A novel isolation method for macrophage-like cells from mixed primary cultures of adult rat liver cells.</a> J. Immunol. Methods. 360, 47-55 (2010).</li><li>Gorrell, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+fibrosis:+the+hepatocyte+revisited.">Liver fibrosis: the hepatocyte revisited.</a> Hepatology. 46, 1659-1661 (2007).</li></ol>

Tous les animaux ont été gardés et gérés à l&#39;animalerie de l&#39;Institut national de santé animale, selon les directives et les règlements énoncés par le Comité de l&#39;institution pour les expérimentations animales.

Nous rapportons ici une méthode simple et efficace d&#39;obtenir des macrophages dans les cultures mixtes primaires de l&#39;adulte les cellules du foie de rat. Notre méthode repose d&#39;abord sur l&#39;activité proliférative roman de macrophages dans la culture mixte de cellules de foie de rat, puis l&#39;isolement et la purification ultérieure de ces cellules sur la base de leurs caractéristiques biologiques comme les macrophages 9. Il peut être possible les macrophages proliféré dans la culture mixte de cellules primaires du foie adulte peut provenir de Kupffer ou des cellules liées qui ont contaminé dans la fraction du parenchyme hépatocytes. Les macrophages pourraient avoir répondu à des changements environnementaux causés par la transformation du parenchyme hépatocytaire 10 et d&#39;autres cellules fibroblastiques dans les cultures mixtes primaires. En conclusion, notre méthode permet d&#39;isoler des cellules de type macrophage, en nombre suffisant et la pureté de la culture primaire de foie de rat en utilisant cellswithout équipements sophistiqués et des compétences techniques. En outre, la procédure d&#39;isolement peut être répétée avec le ballon de la même culture pendant plus de deux semaines. Cette méthode pourrait être applicable à d&#39;autres espèces de mammifères.

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires (optionnel) </th></tr><tr><td> Solution de pentobarbital sodique </td><td> Kyoritsu Seiyaku </td><td> Injection Somnopentyl </td><td></td></tr><tr><td> Hank solution saline équilibrée (HBSS) </td><td> Invitrogen </td><td> 14185-052 </td><td></td></tr><tr><td> L&#39;éthylène glycol tétraacétique (EGTA) </td><td> Sigma-Aldrich </td><td> E-4378 </td><td></td></tr><tr><td> Collagénase </td><td> Wako Pure Chemical Industries, Ltd </td><td> 032-10534 </td><td> Dissociation cellulaire de grade (Lot vérifier au besoin) </td></tr><tr><td> Inhibiteur de la trypsine </td><td> Sigma-Aldrich </td><td> T-9128 </td><td></td></tr><tr><td> Eagle milieu essentiel minimum (MEM) </td><td> Sigma-Aldrich </td><td> M-4655 </td><td></td></tr><tr><td> Modifié par Dulbecco Eagle (DMEM) </td><td> Sigma-Aldrich </td><td> D-6459 </td><td> Haute de glucose de type </td></tr><tr><td> Sérum de veau foetal (FCS) </td><td> HyClone </td><td> SH30070.03 </td><td> Inactivé par la chaleur à 56 ° C pendant 30 min. (Lot vérifier au besoin) </td></tr><tr><td> TrypLE express </td><td> Invitrogen </td><td> 12605 </td><td></td></tr><tr><td> Phosphate de Dulbecco saline tamponnée (PBS) </td><td> Invitrogen </td><td> 14190 </td><td> Ca 2 +-Mg 2 + libre </td></tr><tr><td> β-mercaptoéthanol </td><td> Sigma-Aldrich </td><td> M-7522 </td><td> Stock: 100 mM dans l&#39;eau distillée </td></tr><tr><td> Insuline </td><td> Sigma-Aldrich </td><td> I-5500 </td><td> Stock: 10 mg / ml dans HCl 0,1 N </td></tr><tr><td> Pénicilline / streptomycine </td><td> Invitrogen </td><td> 15070 </td><td></td></tr><tr><td> Tamis cellulaire </td><td> BD Biosciences </td><td> 352360 </td><td> Maillage: 100 um </td></tr><tr><td> Flacon de culture tissulaire </td><td> Sumitomo Bakelite Co., Ltd </td><td> MS-21250 </td><td> Surface: 75 cm 2 </td></tr><tr><td> Des boîtes de culture tissulaire en plastique non- </td><td> BD Biosciences </td><td> 351005 </td><td></td></tr><tr><td> Grattoir à cellules </td><td> Corning Inc </td><td> 3010 </td><td></td></tr><tr><td> Réciproque agitateur </td><td> TAITEC </td><td> NR-1 </td><td></td></tr></tbody></table>

a simple and efficient method to isolate macrophages from mixed primary cultures of adult liver cells

Les cellules de Kupffer sont les macrophages résidents du foie spécifiques et jouent un rôle important dans les fonctions physiologiques et pathologiques du foie 1-3. Bien que les méthodes d&#39;isolement des macrophages du foie ont été bien décrits 4-6, la plupart de ces méthodes nécessitent des équipements sophistiqués, comme une élutriateur centrifuges et des compétences techniques. Ici, nous proposons une nouvelle méthode pour obtenir des macrophages du foie, en nombre suffisant et la pureté du mélange des cultures primaires de cellules de foie de rat adulte, comme illustré schématiquement à la figure 1. Après dissociation des cellules du foie en deux étapes la méthode de perfusion 7,8, une fraction essentiellement composée des hépatocytes parenchymateux est préparée et ensemencée dans T75 flacons de culture tissulaire avec un milieu de culture composé de DMEM et 10% des hépatocytes FCS.Parenchymal perdent la morphologie des cellules épithéliales dans quelques jours dans la culture, dégénèrent ou se transforment en cellules de type fibroblaste (figure 2). Comme le produit de la culture, autour du jour 6, contraste de phase-lumineux, rond cellules de type macrophage commencent à proliférer sur la feuille de cellules fibroblastiques (figure 2). La croissance des cellules de type macrophage poursuivre et atteindre des niveaux maximum autour de 12 jours, couvrant la couche de cellules sur la surface du flacon. En secouant les flacons de culture, les macrophages sont facilement en suspension dans le milieu de culture. Le transfert ultérieur et d&#39;incubation est courte dans le résultat des plats en plastique dans l&#39;adhésion sélective des macrophages (figure 3), où, comme d&#39;autres cellules contaminantes restent suspendues. Après plusieurs rinçages avec du PBS, les macrophages attachés sont récoltés. Plus de 10 6 cellules peuvent être récoltées à plusieurs reprises à partir du même flacon T75 culture de tissus de deux à trois jours d&#39;intervalle pendant plus de deux semaines (figure 3). Les puretés des macrophages isolés étaient de 95 à 99%, évaluée par cytométrie de flux ou immunocytochimie avec des macrophages chez le rat des anticorps spécifiques (figure 4). Les cellules isolées montrent phagocytose active de perles polystylene (figure 5), réponse proliférative à la GM-CSF recombinant, la sécrétion des maladies inflammatoires / anti-inflammatoires des cytokines après stimulation par le LPS, et la formation de cellules géantes multinucléées 9. En conclusion, nous proposons une méthode simple et efficace pour obtenir des macrophages du foie, en nombre suffisant et la pureté sans équipement complexe et skills.This méthode pourrait s&#39;appliquer à d&#39;autres espèces de mammifères.

Watch this Scientific Journal Video about A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells at JoVE.com

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

Une nouvelle méthode pour obtenir des macrophages de culture primaire de cellules de foie de rat est décrit. Cette méthode utilise la prolifération des macrophages dans la culture, suivie par la secousse de flacons de culture et de la purification par l&#39;attachement sélectif plats en plastique. Cette technique fournit efficacement des macrophages du foie, sans équipement complexe et des compétences.

Une méthode simple et efficace pour isoler les macrophages de mixte cultures primaires de cellules hépatiques adultes

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

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Immunology and Infection

37.9K vues.  National Institute of Agrobiological Sciences, Tsukuba, Japan. Une nouvelle méthode pour obtenir des macrophages de culture primaire de cellules de foie de rat est décrit. Cette méthode utilise la prolifération des macrophages dans la culture, suivie par la secousse de flacons de culture et de la purification par l'attachement sélectif plats en plastique. Cette technique fournit efficacement des macrophages du foie, sans équipement complexe et des compétences.

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

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

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

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

Génération des cultures organotypiques de Raft kératinocytes humains primaires

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

Recherche

Immunologie et infection

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

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

Isolement primaire microglies de cultures mixtes de cellules gliales de tissus de cerveau de rat nouveau-né

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Neutrophils are the most abundant leukocytes in peripheral blood. These cells are the first to appear at sites of inflammation and infection, thus becoming the first line of defense against invading microorganisms. Neutrophils possess important antimicrobial functions such as phagocytosis, release of lytic enzymes, and production of reactive oxygen species. In addition to these important defense functions, neutrophils perform other tasks in response to infection such as production of proinflammatory cytokines and inhibition of apoptosis. Cytokines recruit other leukocytes that help clear the infection, and inhibition of apoptosis allows the neutrophil to live longer at the site of infection. These functions are regulated at the level of transcription. However, because neutrophils are short-lived cells, the study of transcriptionally regulated responses in these cells cannot be performed with conventional reporter gene methods since there are no efficient techniques for neutrophil transfection. Here, we present a simple and efficient method that allows detection and quantification of nuclear factors in isolated and immunolabeled nuclei by flow cytometry. We describe techniques to isolate pure neutrophils from human peripheral blood, stimulate these cells with anti-receptor antibodies, isolate and immunolabel nuclei, and analyze nuclei by flow cytometry. The method has been successfully used to detect NF-&kappa;B and Elk-1 nuclear factors in nuclei from neutrophils and other cell types. Thus, this method represents an option for analyzing activation of transcription factors in isolated nuclei from a variety of cell types.

Neutrophils are the most abundant leukocytes in blood. Neutrophils possess transcriptionally regulated functions such as production of proinflammatory cytokines and inhibition of apoptosis. These functions can be studied with the method presented here, which allows detection and quantification of nuclear factors by flow cytometry in isolated nuclei

Une méthode simple et efficace pour détecter l&#39;activation du facteur nucléaire dans les neutrophiles humains par cytométrie en flux

Neutrophils  are the most abundant leukocytes in peripheral blood. These cells are the first  to appear at sites of inflammation and infection, thus ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

La transfection très efficace des droits de THP-1 par les macrophages nucléofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Une méthode de rendement efficace et haute pour l&#39;isolement des souris dendritiques sous-ensembles cellulaires

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

Une approche simple et efficace de construire Mutant Vaccinia vecteurs de virus

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Génération et identification de GM-CSF dérivé alvéolaires comme Macrophages et cellules dendritiques à partir de moelle osseuse de souris

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

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

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

L&#39;induction d&#39;une réponse inflammatoire dans les cultures hépatocytes primaires de souris

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

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is that they may induce traumatic lesions on the surface of the eye, which can progress to scarring, eyelid deformity, limbal stem cell deficiency and in some cases, cause great discomfort to the subject. To avoid these clinical problems, impression cytology (IC) was developed to diagnose dry eye disease and later neoplasia, atopic disease, vernal keratoconjunctivitis and keratoconjunctivitis sicca. Typically, clinicians manually cut filter papers into required shapes and apply these to the ocular surface. Here, we describe how to perform IC using a commercially available medical device. This technique is explained here followed by immunophenotyping by flow cytometry. This technique requires less manual handling and causes less injury to the ocular surface.

The exposed normal ocular surface consists of cornea and conjunctiva. Epithelial cells, goblet cells and immune cells are present in the conjunctiva. Here, a non-invasive, technique of impression cytology is described using an impression cytology device and flow cytometry to analyze immune cells in the conjunctiva.

Une manière non invasive d&#39;isoler et de phénotype des cellules de la conjonctivite

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

Une méthode Simple et efficace pour établir les NK et les lymphocytes T lignes provenant de Patients avec une Infection chronique Active Virus Epstein - Barr

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...