Financé par le programme intra-muros à l&#39;Université en uniforme des services.

1. Dissection des tissus de cerveau de rat nouveau-né <ol><li> Refroidissement Leibovitz L-15 milieux conditionnés (Leibowitz L-15 + 0,1% de BSA + Pen 1% / Strep) à 4 ° C. Milieux de culture chaudes (DMEM + 10% de FBS + 1% de pénicilline / streptomycine) à 37 ° C. Préparer 4 60x15 mm boîtes de Pétri avec 4-5 ml de L-15 climatisées médias sur la glace. Stériliser tous les instruments chirurgicaux avec de l&#39;éthanol 100%. </li><li> Rincer les chiots nouveau-nés P2 rap avec de l&#39;éthanol 70%. (Ratons nouveau-nés âgés de P1 à P5 peut être utilisé dans ce protocole.) </li><li> Rapidement décapiter un raton avec des ciseaux bien aiguisés stériles et déposer la tête immédiatement dans l&#39;éthanol à 70%. Répétez l&#39;opération pour un total de 5 ratons ensuite transférer la tête dans une solution saline. </li><li> Retirez les cerveaux entiers de la tête et les placer dans une boîte de Pétri avec 4 - 5 ml solution L-15 (Leibowitz L-15 + 0,1% de BSA + 1% Pen / Strep) sur la glace. </li><li> Répéter les procédures 1.2 - 1.4 pour les chiots qui restent dans la litière. </li><li> Retirez les méninges du cerveau et de transférer le destissu cérébral IRED (c.-à-cortex, le cervelet, le cerveau entier, etc) dans un nouveau plat de Pétri avec 4-5 ml de solution de L-15 (Leibowitz L-15 + 0,1% de BSA + Pen 1% / Strep) sur la glace. </li></ol> 2. Préparation de la population de cellules gliales mixte <ol><li> Sans endommager le tissu cérébral avec des ciseaux ou une lame, transférer le tissu avec une pipette de 10 ml à 50 ml une stérile tube conique. </li><li> Centrifuger conique à 2500 RCF pendant 5 min à 4 ° C. </li><li> Aspirer le surnageant. Ajouter 4-5 ml de frais L-15 médias (Leibowitz L-15 + 0,1% + 1% BSA Pen / Strep). </li><li> Introduire à la pipette des tissus de haut en bas 10 fois avec une pipette stérile de 10 ml. </li><li> Placer un tamis cellulaire (100 pores um) sur un nouveau tube de 50 ml conique. Tissus pipette de haut en bas une fois avec une solution stérile de pipette 5 ml et que la pipette au ras de la crépine cellule, distribuer le matériau à travers la crépine cellule dans le tube conique. </li><li> Rincer la crépine cellule avec 4-5 ml de frais L-15 médias (Leibowitz L-15 + 0,1% BSUn stylo + 1% / Strep). </li><li> Centrifuger conique avec des cellules tendues à 2500 RCF pendant 5 min à 4 ° C. </li></ol> 3. Placage et d&#39;entretien des cultures mixtes de cellules gliales <ol><li> Pour chaque cerveau de rat nouveau-nés traités, préparer 1 T-75 stériles flacon en ajoutant 12 ml de milieux de culture (DMEM + 10% de FBS + 1% de pénicilline / streptomycine) dans chaque flacon. </li><li> Aspirer le surnageant des cellules granulées et ajouter 5-6 ml de milieux de culture pour le culot cellulaire. Introduire à la pipette de haut en bas 10 fois avec une pipette de 10 ml. </li><li> Transférer un volume égal de suspension cellulaire dans chaque fiole T-75. </li><li> Incuber fioles dans un 5% de CO 2 incubateur à 37 ° C pour un total de 1-3 semaines. </li><li> Flacons devrait d&#39;abord être incubées pendant 5 jours, et le cinquième jour, remplacer les milieux de culture dans chaque flacon avec 12 ml de milieu frais et retourner à l&#39;incubateur. Puis, tous les 3 jours, remplacer les milieux conditionnés dans chaque flacon avec 12 ml de milieu frais pour atteindre la confluence. Ce doit être le fairene très attentivement sans toucher le fond des flacons où les cellules se fixent. </li></ol> 4. Isolement et Revêtement de l&#39;enseignement primaire microglies <ol><li> Après mixtes cultures gliales sont complètement confluentes, retirez flacons de l&#39;incubateur et les caches flacon avec du parafilm pour éviter les échanges gazeux avec l&#39;air ambiant. </li><li> Agiter flacons à 100 RPS (Companion Lab SI-600, JEIO Tech) pendant 1 heure à 37 ° C. </li><li> Recueillir les médias de tous les flacons avec une pipette de 10 ml, sans perturber la couche d&#39;astrocytes à la surface ballon. Placez le support dans des tubes de 50 ml coniques. Ajouter des milieux frais aux flacons et bouteilles de retour à l&#39;incubateur. </li><li> Centrifuger les tubes coniques à 2500 RCF pendant 5 min à 4 ° C. </li><li> Aspirer surnageant de tous les tubes coniques. Les culots cellulaires sont élevés cellules de la microglie de pureté. Pellets Remettre en suspension dans 1 ml de placage microglie médias (DMEM + FES 10% + 1% de pénicilline / streptomycine). </li><li> Comptez la densité cellulaire dans les médias remises en suspension à l&#39;aideun hémocytomètre. </li><li> Ajouter un volume approprié de géloses microglies pour atteindre un 2 x 10E5 cellules par ml de densité. Plate comme il convient pour l&#39;analyse expérimentale. </li><li> Autoriser les microglies pour attacher la nuit. </li></ol> 5. Les résultats représentatifs Le protocole décrit ci-dessus les résultats dans les cultures primaires de haute pureté microglie. Comme déterminé par immunohistochimie pour un marqueur cellulaire de macrophages / microglies spécifique (Iba1), plaqués cultures microglies sont&gt; à 90% (figure 2). En outre, la coloration des marqueurs de cellules astrocytes, oligodendrocytes et des neurones spécifiques démontre une contamination minimale (figure 2). Après avoir établi mixtes cultures de cellules gliales, la microglie peut être isolé en secouant jusqu&#39;à 4 fois successives. L&#39;isolement initial microglie donnera environ 2,2 x 106 cellules / ml ou environ 9 millions de cellules par 10 flacons et le rendement attendu diminue à chaque sha successifske. La microglie isolés par cette méthode ont été utilisées avec succès pour étudier la capacité de phagocytose, la fonction, comme la production d&#39;oxyde nitrique, et la toxicité neuronale (Figure 3) 13-14. <img alt="Figure 1" src="/files/ftp_upload/3814/3814fig1.jpg" /> Figure 1. Vue d&#39;ensemble du protocole de préparation de cultures mixtes de cellules gliales et l&#39;isolement de la microglie primaire. <img alt="Figure 2" src="/files/ftp_upload/3814/3814fig2.jpg" /> Figure 2. Isolement de la microglie de haute pureté tel que vérifié par une analyse immunohistochimique utilisant la microglie (Iba-1, rouge), des astrocytes (GFAP, vert), neurone (NeuN, rouge) et des oligodendrocytes (CC1, rouge) des marqueurs spécifiques. La coloration de l&#39;ADN des cellules en culture par DAPI (en bleu) est également indiquée. <img alt="Figure 3" src="/files/ftp_upload/3814/3814fig3.jpg" /> Figure 3. Microglie isolés dans la descri méthodelit ont été utilisées dans un certain nombre de tests, y compris, mais sans s&#39;y limiter, les dosages, les dosages de la fonction de phagocytose et des études de neurotoxicité. Dans ces études, nous avons constaté que la microglie (Iba-1 positive, vert), lorsqu&#39;ils sont exposés à contrôler (A) ou LPS (B) des médias traités, augmenter leur phagocytose de billes de latex marquées par fluorescence (rouge), qui peuvent être quantifiées ( C). En outre, le LPS (1ng/ml) le traitement peut augmenter la production d&#39;oxyde nitrique dans la microglie (D). Enfin, via à la fois transwell insert-séparés ou directs microglie-neurones cultures, nous avons constaté que les microglies incubées avec LPS peuvent induire la mort des cellules neuronales, telle que mesurée par la lactate déshydrogénase (LDH) de libération (E, F).

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	<li>Ransohoff, R. M., Perry, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+physiology:+unique+stimuli,+specialized+responses.">Microglial physiology: unique stimuli, specialized responses.</a> Annu. Rev. Immunol. 27, 119-145 (2009).</li><li>Raivich, G., Bohatschek, M., Kloss, C. U., Werner, A., Jones, L. L., Kreutzberg, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroglial+activation+repertoire+in+the+injured+brain:+graded+response,+molecular+mechanisms+and+cues+to+physiological+function.">Neuroglial activation repertoire in the injured brain: graded response, molecular mechanisms and cues to physiological function.</a> Brain Res. Brain Res. Rev. 30, 77-105 (1999).</li><li>Loane, D. J., Byrnes, K. R. <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+microglia+in+neurotrauma.">Role of microglia in neurotrauma.</a> Neurotherapeutics. 7, 245-265 (2010).</li><li>Glass, C. K., Saijo, K., Winner, B., Marchetto, M. C., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+underlying+inflammation+in+neurodegeneration.">Mechanisms underlying inflammation in neurodegeneration.</a> Cell. 140, 918-934 (2010).</li><li>Pocock, J. M., Liddle, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+signalling+cascades+in+neurodegenerative+disease.">Microglial signalling cascades in neurodegenerative disease.</a> Prog. Brain Res. 132, 555-565 (2001).</li><li>Block, M. L., Hong, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+microglial+activation+and+progressive+dopaminergic+neurotoxicity.">Chronic microglial activation and progressive dopaminergic neurotoxicity.</a> Biochem. Soc. Trans. 35, 1127-1132 (2007).</li><li>Barger, S. W., Basile, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+microglia+by+secreted+amyloid+percursor+protein+evokes+release+of+glutamate+by+cystine+exchange+and+attenuates+synpatic+function.">Activation of microglia by secreted amyloid percursor protein evokes release of glutamate by cystine exchange and attenuates synpatic function.</a> J. Neurochem. 76, 846-854 (2001).</li><li>Ni, M., Aschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+rat+primary+microglia:+isolation,+culturing,+and+selected+applications.">Neonatal rat primary microglia: isolation, culturing, and selected applications.</a> Curr. Protoc. Toxicol. Chapter 12, Unit 12.17 (2010).</li><li>Hassan, N. F., Rifat, S., Campbell, D. E., McCawley, L. J., Douglas, S. D. <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+flow+cytometric+characterization+of+newborn+mouse+brain-derived+microglia+maintained+in+vitro.">Isolation and flow cytometric characterization of newborn mouse brain-derived microglia maintained in vitro.</a> J. Leukoc. Biol. 50, 86-92 (1991).</li><li>Hassan, N. F., Prakash, K., Chehimi, J., McCawley, L. J., Douglas, S. D. <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+characterization+of+newborn+rabbit+brain-derived+microglia.">Isolation and characterization of newborn rabbit brain-derived microglia.</a> Clin. Immunol. Immunopathol. 59, 426-435 (1991).</li><li>Hassan, N. F., Campbell, D. E., Rifat, S., Douglas, S. D. <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+characterization+of+human+fetal+brain-derived+microglia+in+in+vitro+culture.">Isolation and characterization of human fetal brain-derived microglia in in vitro culture.</a> Neuroscience. 41, 149-158 (1991).</li><li>Frank, M. G., Wieseler-Frank, J. L., Watkins, L. R., Maier, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isolation+of+highly+enriched+and+quiescent+microglia+from+adult+rat+hippocampus:+immunophenotypic+and+functional+characteristics.">Rapid isolation of highly enriched and quiescent microglia from adult rat hippocampus: immunophenotypic and functional characteristics.</a> J. Neurosci. Methods. 151, 121-130 (2006).</li><li>Byrnes, K. R., Stoica, B., Loane, D. J., Riccio, A., Davis, M. I., Faden, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabotropic+glutamate+receptor+5+activation+inhibits+microglial+associated+inflammation+and+neurotoxicity.">Metabotropic glutamate receptor 5 activation inhibits microglial associated inflammation and neurotoxicity.</a> Glia. 57, 550-560 (2009).</li><li>Loane, D. J., Stoica, B. A., Pajoohesh-Ganji, A., Byrnes, K. R., Faden, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+metabotropic+glutamate+receptor+5+modulates+microglial+reactivity+and+neurotoxicity+by+inhibiting+NADPH+oxidase.">Activation of metabotropic glutamate receptor 5 modulates microglial reactivity and neurotoxicity by inhibiting NADPH oxidase.</a> J. Biol. Chem. 284, 15629-15639 (2009).</li></ol>

Pas de conflits d&#39;intérêt déclarés.

Alors que ce protocole est régulièrement utilisé pour produire la microglie pur et sain pour les expériences de recherche, un examen attentif des aspects techniques au cours de parties critiques de la procédure permettra de minimiser la variabilité dans la microglie isolée. Tout d&#39;abord, lors de la dissection du tissu cérébral de rats nouveau-nés petits, travaillant en temps opportun est nécessaire pour minimiser les dommages hypoxiques et ischémiques du tissu. Cependant, il est également important d&#3...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Le numéro de catalogue </td><td> Commentaires </td></tr><tr><td> 60 mm x 15 mm boîtes de Pétri </td><td> Fisher marque </td><td> 0875713A </td><td></td></tr><tr><td> De Sharp ciseaux de dissection </td><td> Outils belle science </td><td> 14094-11 </td><td></td></tr><tr><td> Dumont # 7b pince standard des conseils, des courbes, 11cm </td><td> Outils belle science </td><td> 11270-20 </td><td></td></tr><tr><td> Dumont # 5 pince standard des conseils, des droites, 11 cm </td><td> Outils belle science </td><td> 11251-10 </td><td></td></tr><tr><td> Tubes de 50 ml à centrifuger coniques </td><td> VWR </td><td> 89039-656 </td><td></td></tr><tr><td> 5 pipettes sérologiques ml </td><td> Grenier Bio One </td><td> 606180 </td><td></td></tr><tr><td> 10 pipettes sérologiques ml </td><td> Grenier Bio One </td><td> 607180 </td><td></td></tr><tr><td> 100 um crépine stérile cellule de nylon </td><td> Faucon </td><td> 35-2360 </td><td></td></tr><tr><td> 75 cm 2 flacons de culture tissulaire </td><td> Corning </td><td> 430641 </td><td></td></tr><tr><td> Dulbecco milieu essentiel minimal (DMEM) </td><td> Gibco (Invitrogen) </td><td> 31053-028 </td><td></td></tr><tr><td> Leibovitz milieu L-15 </td><td> Gibco (Invitrogen) </td><td> 11415064 </td><td></td></tr><tr><td> Sérum de veau fœtal </td><td> Gibco (Invitrogen) </td><td> 16000-036 </td><td></td></tr><tr><td> Fœtale sérum équin </td><td> Pêcheur </td><td> SH3007402 </td><td></td></tr><tr><td> La pénicilline-streptomycine </td><td> Gibco (Invitrogen) </td><td> 15140163 </td><td></td></tr><tr><td> L&#39;éthanol à 100% </td><td> La Société Warner Graham </td><td> 64-17-5 </td><td></td></tr><tr><td> Phosphate solution saline tamponnée, 10X, pH 7,4 </td><td> Qualité biologique, inc. </td><td> 119-069-131 </td><td> 1X dans l&#39;eau distillée stérile </td></tr><tr><td> Sacs de Biohazard </td><td> VWR </td><td> 14220-028 </td><td></td></tr><tr><td> Hémocytomètre </td><td> Hausser scientifique </td><td> 1492 </td><td></td></tr><tr><td> Des plaques 6 puits culture cellulaire avec une surface CellBIND </td><td> Corning </td><td> 3335 </td><td></td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue at JoVE.com

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

Isoler les microglies primaire à partir de l&#39;hétérogénéité cellulaire du cerveau est essentielle pour enquêter sur leur rôle dans les deux conditions physiologiques et pathologiques. Ce protocole décrit une isolation mécanique et technique mixte de culture cellulaire qui fournit un rendement élevé et de haute pureté, viables primaires pour les cellules microgliales<em&gt; In vitro</em&gt; Étude et les applications en aval.

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 ...

primary-microglia-isolation-from-mixed-glial-cell-cultures-neonatal

Research

JoVE Journal

Immunology and Infection

39.1K vues.  Uniformed Services University. Isoler les microglies primaire à partir de l'hétérogénéité cellulaire du cerveau est essentielle pour enquêter sur leur rôle dans les deux conditions physiologiques et pathologiques. Ce protocole décrit une isolation mécanique et technique mixte de culture cellulaire qui fournit un rendement élevé et de haute pureté, viables primaires pour les cellules microgliales In vitro Étude et les applications en aval.

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

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

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

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 ...

Recherche

Immunologie et infection

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 ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Microglia de culture du système nerveux central néonatale et de l&#39;adulte

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-&#954;B (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-&#945; (TNF-&#945;), upon lipopolysaccharide (LPS) and Pam3CSK4 (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions.

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (Pam).

Isolation des corticale microglie avec immunophénotype préservée et fonctionnalité De murins nouveau-nés

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

Isolement et injection intraveineuse de moelle osseuse de souris dérivées monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of puberty - more than ten years after the establishment of systemic immune tolerance. Spermatogenic cells express a number of proteins that may be seen as non-self by the immune system. The testis must then be able to establish tolerance to these neo-antigens on the one hand but still be able to protect itself from infections and tumor development on the other hand. Therefore the testis is one of a few immune privileged sites in the body that tolerate foreign antigens without evoking a detrimental inflammatory immune response. Sertoli cells play a key role for the maintenance of this immune privileged environment of the testis and also prolong survival of cotransplanted cells in a foreign environment. Therefore primary Sertoli cells are an important tool for studying the immune privilege of the testis that cannot be easily replaced by established cell lines or other cellular models. Here we present a detailed and comprehensive protocol for the isolation of Sertoli cells - and peritubular cells if desired - from rat testes within a single day.

Primary Sertoli cells are required for studying testis-immune privilege, signal transduction during inflammation or infection, and utilization of their immunoprotective properties. Here we describe an enzyme-based protocol for the isolation of highly purified primary Sertoli cells and peritubular cells from rat testes.

Isolement des cellules de Sertoli et péritubulaire cellules de rat Testicules

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of ...

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 ...

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 ...

Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After First Colostrum Feeding

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression of proteins known to respond to metabolic stress was measured in brain-nuclei isolates using Western blotting. This was done to assess whether metabolic stress-induced nutrient insufficiency in the body triggered neuronal stress. We have previously demonstrated that nutrient insufficiency in neonates elicits metabolic stress in the gut. Furthermore, colostrum oxytocin modulates cellular stress response, inflammation, and autophagy markers in newborn rat gut villi prior to and after first feed. Signaling protein markers associated with the endoplasmic reticulum stress [ER chaperone binding immunoglobulin protein (BiP), eukaryotic translation initiation factor 2A (eIF2a), and eIF2a kinase protein kinase R (p-PKR)], as well as two inflammation-signaling proteins [nuclear factor-&#954;B (NF-kB) and inhibitor &#954;B (IkB)], were measured in newborn brain nuclei [nucleus of the solitary tract (NTS), paraventricular nucleus (PVN), supra-optic nucleus (SON), cortex (CX), striatum nuclei (STR), and medial preoptic nucleus (MPO)] before the first feed (unprimed by colostrum) and after the start of nursing (primed by colostrum). Expression of BiP/GRP78 and p-eIF2a were upregulated in unprimed and downregulated in primed NTS tissue. NF-kB was retained (high) in the CX, STR, and MPO cytoplasm, whereas NF-kB was lower and unchanged in NTS, PVN, and SON in both conditions. The collective BiP and p-eIF2 findings are consistent with a stress response. eIf2a was phosphorylated by dsRNA dependent kinase (p-PKR) in the SON, CX, STR, and MPO. However, in the NTS (and to a lesser extent in PVN), eIf2a was phosphorylated by another kinase, general control nonderepressible-2 kinase (GCN2). The stress-modulating mechanisms previously observed in newborn gut enterocytes appear to be mirrored in some OTR-rich brain regions. The NTS and PVN may utilize a different phosphorylation mechanism (under nutrient deficiency) from other regions and be refractory to the impact of nutrient insufficiency. Collectively, this data suggests that brain responses to nutrient insufficiency stress are offset by signaling from colostrum-primed enterocytes.

Here, we present a protocol to isolate brain nuclei in the neonatal rat brain in conjunction with first colostrum feeding. This technique allows the study of nutrient insufficiency stress in the brain as modulated by enterocyte signaling.

Évaluer le Stress cellulaire et l’Inflammation dans les noyaux en cerveau sécrétant ocytocine discrète chez le Rat néonatal avant et après la première tétée de Colostrum

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression ...

Concomitant Isolation of Primary Astrocytes and Microglia for Protozoa Parasite Infection

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central nervous system (CNS). The increasing evidences of their involvement in the control of infectious diseases justify the emerging interest in the improvement of methodologies to isolate primary astrocytes and microglia in order to evaluate their responses to infections that affect the CNS. Considering the impact of Trypanosoma cruzi (T. cruzi) and Toxoplasma gondii (T. gondii) infection in the CNS, here we provide a method to extract, maintain, dissociate and infect murine astrocytes and microglia cells with protozoa parasites. Extracted cells from newborn cortices are maintained in vitro for 14 days with periodic differential media replacement. Astrocytes and microglia are obtained from the same extraction protocol by mechanical dissociation. After phenotyping by flow cytometry, cells are infected with protozoa parasites. The infection rate is determined by fluorescence microscopy at different time points, thus enabling the evaluation of differential ability of glial cells to control protozoan invasion and replication. These techniques represent simple, cheap and efficient methods to study the responses of astrocytes and microglia to infections, opening the field for further neuroimmunology analysis.

The overall goal of this protocol is to instruct how to extract, maintain, and dissociate murine astrocyte and microglia cells from the central nervous system, followed by infection with protozoa parasites.

Isolement concomitant des astrocytes primaires et des microglies pour l’infection de parasite de Protozoa

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central ...