Ce travail a été soutenu par NIEHS R01ES014470 (KMZ).

Avant de commencer ce protocole, recueillir des souriceaux nouveau-nés à jour postnatal 1-3 (P1-3) dans un récipient stérile imbriquée avec literie de cage d&#39;origine pour la protection et la chaleur. Il est important de travailler rapidement et efficacement à travers ce protocole pour optimiser le rendement de la microglie. S&#39;il vous plaît voir le tableau 1 pour la liste complète des réactifs. Une. Préparation d&#39;instruments, Culture Media, et plats <ol><li> Préparer 10 ml / pup microglie complet Support (MCM; 1x Minimal Essential Medium Earle [MEM] supplémenté avec 1 mM de L-glutamine, du pyruvate de sodium 1 mM, 0,6% v / v de D-(+)-glucose, 100 pg / ml pénicilline / streptomycine (P / S), 4% v / v de sérum bovin fœtal (FBS), 6% v / v de sérum de cheval), et ajouter à chaque flacon T-75 (1 flacon / chiot); bouchon et le lieu horizontalement dans un incubateur (37 ° C, 5% CO 2) à s&#39;équilibrer pendant 1 heure. Il est indispensable à l&#39;équilibre de ce support dans la fiole (s) avant de commencer la dissection. </li><li> Prepare, aliquote, et chaude en 37 ° C MCM bain d&#39;eau pour la dissection (6 ml / chiot) et dans un tube conique de MCM distinct pour la remise en suspension de cellules (2 ml / chiot). Préparer, aliquote, et le froid sur la glace Mincing Media (MM; HBSS complété par: 100 pg / ml P / S, 0,01 M HEPES; 3 ml / chiot), et de dissection Media (DM; MEM supplémenté avec 100 pg / ml P / S; 2 ml / chiot). </li><li> Stériliser les instruments chirurgicaux suivants par immersion dans de l&#39;éthanol à 70% pendant 10 min: ciseaux # 1 (1; décapiter), pince # 1 (2; d&#39;assurer le nez et supprimer cortex), ciseaux # 2 (1; non en biais; d&#39;ouvrir cuir chevelu ), pince # 2 (1; d&#39;enlever le cuir chevelu), pince # 3 (1; d&#39;enlever crâne), pince # 4 (2; d&#39;enlever méninges), ciseaux # 3 (1; pour hacher le tissu cérébral). Laisser sécher à l&#39;air sur une compresse stérile. </li><li> Essuyez l&#39;horizontale aéré poste de travail et la dissection microscope avec 70% d&#39;éthanol. </li><li> Distribuer DM en 2, 100 mm boîtes de Pétri stériles pour capturer la tête (3 ml; 1 plat / 3 chiots) et la dissection (1 ml;1 plat / chiot; refroidi sur la glace avant la dissection). Distribuer MM dans une stérile, 100 mm boîte de Petri pour hacher le tissu cérébral (1 ml; 1 plat / chiot). </li></ol> 2. Dissection de tissu cérébral <ol><li> Avant l&#39;euthanasie, désinfecter doucement le cou et la tête du chiot avec 70% d&#39;éthanol en utilisant un tissu de laboratoire. Avec des ciseaux # 1, euthanasier rapidement chiot en enlevant la tête; tête de capture en boîte de Pétri préalablement préparé. Euthanasier et compléter la dissection d&#39;un chiot avant de procéder à l&#39;animal suivant. </li><li> Transférer la tête dans le plat de dissection froide; sous microscope de dissection fixer la tête en pinçant le nez à l&#39;aide pince # 1 (sinon, la tête peut être fixée en perçant la pince à travers la cavité de l&#39;œil); cuir chevelu ouvert à l&#39;aide de ciseaux # 2 par la création d&#39;un Y-cut: commencer à partir de base de la tête et à un angle vers les yeux. Utilisez pince # 3 pour enlever délicatement le cuir chevelu en enlevant latéralement, exposer le crâne. </li><li> Utilisation pince # 3, retirez délicatement le tissu conjonctif à la base detête. Lorsque la base du crâne est exposé crâne soigneusement ouvert en enfilant un bras de pince entre le cerveau et le crâne (la suite de la fissure interhémisphérique); adhérence; tirez doucement vers le haut du crâne à déchirer. </li><li> Broyer le tissu conjonctif sous la face ventrale du cerveau à l&#39;aide pince n ° 1, puis retirez cerveau en utilisant le même outil. Soulevez le cerveau de crâne à l&#39;aide d&#39;une force vers le haut sous la partie ventrale du cerveau. </li><li> Gardez le cerveau dans sa position anatomique: la face dorsale vers le haut pour visualiser les cortex sous le microscope de dissection; garantir le cerveau en place en insérant des conseils de pince à l&#39;intersection du mésencéphale et du cortex; supprimer complètement méninges corticales avec pince n ° 4, puis passez à supprimer les méninges ventrales. Une fois les méninges ont été complètement enlevés, séparer les cortex du reste du cerveau à l&#39;aide pince # 4., Il est essentiel d&#39;éliminer complètement tissu méningé pour maximiser la pureté de la microglie et yield. </li></ol> 3. Brain Tissue Mincing <ol><li> cortex de transfert précédemment préparés hacher plat; continuer à hacher étape utilisant une technique stérile au Cabinet de classe II de sécurité biologique. </li><li> Tissu Mince de cerveau à l&#39;aide de ciseaux # 3; transfert tissu haché dans un tube de 15 ml conique;. Rincer ciseaux et hacher plat avec 1 ml de MM chacun, recueillir et distribuer des rinçages dans le tube de 15 ml conique contenant le tissu haché Cette étape de rinçage maximiser le rendement de la microglie en s&#39;assurant que tout le tissu résiduel est transféré dans le tube conique. Laissez suspension de tissu non perturbée pendant 1-2 min, permettant tissu haché de régler au fond du tube. Ne pas dépasser 2 min que MM ne contient pas de nutriments essentiels. </li><li> Retirez délicatement et jeter 2 ml de surnageant avec générique 1000 pi micropipette avec une pointe de pipette standard, veillant à ne pas perturber le haché tissu réglé. </li&gt; <li> Ajouter 3 ml de MCM au-tissu haché; triturer le tissu avec force en utilisant un générique 1000 pi micropipette avec une pointe de pipette la norme n ° discernable tissu solide doit rester quand trituration est terminée.. Ajouter un autre 3 ml de MCM avec la même pointe de la pipette, la collecte de l&#39;échantillon trituré résiduelle de la pointe de la pipette. </li></ol> 4. La culture de cellules corticales <ol><li> Centrifugeuse le tissu haché pour sédimenter les cellules (215 xg, 5 min; RT; centrifugeuse clinique avec balancement seau rotor). Retour culot de cellules de l&#39;enceinte de sécurité biologique, enlever et jeter le surnageant; remettre en suspension doucement le culot cellulaire (2 ml / tube; MCM), ajouter la remise en suspension des cellules de la préalablement préparé flacon T-75; plafonner le ballon et le placer horizontalement dans un humidifiée, norme incubateur de culture de tissu (à 37 ° C, 5% CO 2). Laisser les cellules incuber pendant 24 heures. </li><li> Après 24 h, tapotez doucement ballon contre paume de la main pour perturberdébris de tissu. Voir les cellules avant et après tapotement pour assurer une élimination complète du débris. Set ballon vertical; enlever et jeter les médias; ajouter MCM frais au fond du flacon (12 ml; 37 ° C); bouchon et remettez-les dans l&#39;incubateur, horizontalement. </li><li> Vérifiez cellules quotidien de contamination et pour surveiller la croissance. </li><li> A environ 17-21 jours in vitro (DIV), les cellules microgliales seront prêts pour la récolte de la culture des cellules gliales mixtes. Pour déterminer si les cellules sont prêtes, examiner les cellules sous un microscope à contraste de phase inversée. Microglies sont prêtes pour la récolte quand à ~ 40% de confluence. Ils apparaissent comme de petites cellules, lumineux, sphériques, de plus en plus comme une monocouche sur d&#39;autres cellules gliales. </li></ol> 5. Isolation des microglies de mixte gliales Culture <ol><li> Pour isoler la microglie, frappez vigoureusement ballon contre un banc de laboratoire. Cette agitation permet la microglie séparées des autres cellules gliales en raison des propriétés de faible adhérence de la microglie. </li><li&gt; Joint T-75 casquettes de ballon avec Parafilm et faire tourner les cultures mixtes gliales (200 rpm; 37 ° C, 5 h). utilisant un agitateur rotatif nonhumidified température contrôlée Ne pas laisser les cellules de secouer pendant plus de 5 heures d&#39;inspecter visuellement. que les microglies ont soulevé la surface du flacon après 5 h, à l&#39;aide d&#39;un microscope à contraste de phase inversée. microglies sont lumineuses, sphérique, flottant cellules. Il y aura une couche résiduelle d&#39;astrocytes et les oligodendrocytes qui adhèrent au fond de la fiole. </li><li> Recueillir milieu surnageant contenant la microglie dans un tube à centrifuger conique stérile; microglie de granulés (215 xg, 5 min; balancer seau rotor). </li><li> Remettre en suspension le culot de la microglie dans chaud microglie du milieu de croissance (MGM; MEM supplémenté avec: pyruvate 1 mM de sodium, 0,6% v / vglucose, 1 mM de L-glutamine, 100 pg / ml de P / S, 5% v / v de FBS; ~ 2 ml / flacon), déterminer la concentration des cellules en utilisant un hémocytomètre, et la plaque 1 x 10 5 cellules / puits dans un format de plaque à 24 puits surplastique ou bordereaux stériles de couverture de verre (500 pl / puits; MGM). le rendement de la microglie moyenne utilisant cette procédure est de 7,5 x 10 5 cellules / chiot cortex. </li><li> 24 heures après placage, supprimer tous les médias et les cellules réalimentation avec frais MGM (37 ° C) pour éliminer les cellules et les débris flottants. Cette étape est essentielle à la survie cellulaire et de maintenir la microglie au repos. Les cellules peuvent être utilisés pour l&#39;expérimentation immédiatement après réalimentation. </li></ol> 6. Exemple Traitements <ol><li> Exposer les cellules à 10 ng / ml de Pam 3 CSK 4 (Pam), 10 ng / ml de lipopolysaccharide (LPS) ou le contrôle du véhicule; incuber pendant 2 ou 24 heures dans un incubateur humidifié (37 ° C, 5% de CO 2). </li></ol> 7. Analyser microglies pour la fonctionnalité Ex vivo par immunofluorescence imagerie et Immunocytochemistry <ol><li> Après traitement, on récolte les cellules des milieux de culture dans des tubes de microcentrifugation (1,5 ml) et centrifuge utilisant une micro de table (2 min; 900 xg) pour effacer les médias. Transférer le surnageant dans un tube de centrifugeuse propre; dosage immédiatement, ou conserver à -20 ° C jusqu&#39;au moment de servir. Analyser la concentration de TNF-α dans les cellules du surnageant de culture par ELISA kit disponible dans le commerce de la souris de TNF-α. </li><li> Laver les cellules avec du tampon phosphate salin (PBS), et fixer les cellules avec 4% (p / v) de paraformaldéhyde / 4% (p / v) de sucrose / PBS pH 7,4 (PFA 4%, 15 min; RT). Après la fixation, laver les cellules avec du PBS pendant 5 min en agitant doucement sur un agitateur orbital. <ol><li> Immunofluorescence / immunocytochimie. Après fixation et de lavage PBS, processus microglies pour immunocytochimie. Toutes les étapes effectuées en agitant doucement. Perméabiliser les cellules dans PBS contenant 0,1% de triton X-100, des cellules de bloc dans PBS/10% de sérum de chèvre normal pendant une heure. Incuber les cellules O / N à 4 ° C avec des anticorps de lapin anti-NF-kB (sous-unité p65, 1/1, 250) ou de lapin anti-anticorps Iba-1 (1/750) dans du tampon de blocage. Visueliser complexe antigène: anticorps après incubation avec fluorescence chèvre anti-lapin conjugué anticorps IgG secondaire (1 h; 1/1, 000). Éliminer l&#39;anticorps secondaire non lié par lavage avec du PBS contenant 0,1% de triton X-100 (3 x 5 min). </li><li> Cellules contre des taches avec 4 &#39;,6-Diamidino-2-phénylindole / PBS (DAPI; 0,1 pg / ml). Afin de visualiser le noyau de la cellule Tous les microglies provenant de CX3CR1-GFP + / - souris expriment la GFP. Utiliser un filtre de longueur d&#39;onde de 350 nm pour visualiser le DAPI, un filtre de longueur d&#39;onde 488 nm pour visualiser la GFP, et un filtre de longueur d&#39;onde 594 nm pour visualiser p65 et Iba-1 immuns. </li></ol></li><li> Monter le couvercle glisse en utilisant une solution aqueuse de montage et de visualiser la microglie en utilisant la microscopie à fluorescence. </li></ol>

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M. <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+murinemicroglial+cells+for+RNA+analysis+or+flow+cytometry.">Isolation of murinemicroglial cells for RNA analysis or flow cytometry.</a> Nat. Protoc. 1, 1947-1951 (2006).</li><li>Ford, A. L., Goodsall, A. L., Hickey, W. F., Sedgwick, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+adult+ramified+microglia+separated+from+other+central+nervous+system+macrophages+by+flow+cytometric+sorting.+Phenotypic+differences+defined+and+direct+ex+vivo+antigenpresentation+to+myelin+basic+protein-reactive+CD4++T+cells+compared.">Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting. Phenotypic differences defined and direct ex vivo antigenpresentation to myelin basic protein-reactive CD4+ T cells compared.</a> J. Immunol. 154, 4309-4321 (1995).</li><li>Ford, A. L., Foulcher, E., Goodsall, A. L., Sedgwick, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+digestion+with+dispase+substantially+reduces+lymphocyte+and+macrophage+cell-surface+antigen+expression.">Tissue digestion with dispase substantially reduces lymphocyte and macrophage cell-surface antigen expression.</a> J. Immunol. Methods. 194, 71-75 (1996).</li><li>Pino, P. A., Cardona, A. E. <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+brain+and+spinal+cord+mononuclear+cells+using+percoll+gradients.">Isolation of brain and spinal cord mononuclear cells using percoll gradients.</a> J. Vis. Exp. , (2011).</li><li>Veremeyko, T., Starossom, S. C., Weiner, H. L., Ponomarev, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+microRNAs+in+microglia+by+real-time+PCR+in+normal+CNS+and+during+neuroinflammation.">Detection of microRNAs in microglia by real-time PCR in normal CNS and during neuroinflammation.</a> J. Vis. Exp. , (2012).</li><li>Su, X., Federoff, H. J., Maguire-Zeiss, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutant+alpha-synuclein+overexpression+mediates+early+proinflammatory+activity.">Mutant alpha-synuclein overexpression mediates early proinflammatory activity.</a> Neurotox. Res. 16, 238-254 (2009).</li><li>Jung, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+fractalkine+receptorCX(3)CR1+function+by+targeted+deletion+and+green+fluorescent+protein+reporter+gene+insertion.">Analysis of fractalkine receptorCX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion.</a> Mol. Cell. Biol. 20, 4106-4114 (2000).</li><li>Imai, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+molecular+characterization+of+fractalkine+receptor+CX3CR1,+which+mediates+both+leukocyte+migration+and+adhesion.">Identification and molecular characterization of fractalkine receptor CX3CR1, which mediates both leukocyte migration and adhesion.</a> Cell. 91, 521-530 (1997).</li><li>Cardona, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+microglial+neurotoxicity+by+the+fractalkine+receptor.">Control of microglial neurotoxicity by the fractalkine receptor.</a> Nat. Neurosci. 9, 917-924 (2006).</li><li>Streit, W. J., Walter, S. A., Pennell, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+microgliosis.">Reactive microgliosis.</a> Prog. Neurobiol. 57, 563-581 (1999).</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></ol>

Les auteurs déclarent que la recherche a été menée en l&#39;absence de relations commerciales ou financières qui pourraient être interprétées comme un conflit d&#39;intérêts potentiel.

La présente procédure offre une méthode efficace pour l&#39;isolement de la microglie corticaux de souris néonatales. Cette procédure a un double avantage de 1) la préservation immunophénotype microglie et la fonctionnalité, telle que déterminée par l&#39;imagerie de fluorescence, immunocytochimie, et ELISA, et 2) permettant la microglie à échéance à la présence d&#39;autres cellules gliales (astrocytes et oligodentrocytes) avant l&#39;isolement, ce qui peut faciliter les interactions cellule-cellule impo...

Cellules de la microglie, les macrophages de surveillance du parenchyme du système nerveux central, représentent environ 12% de la population cellulaire totale du cerveau des mammifères adultes. Les microglies sont issus de la vésicule ombilicale myéloïdes cellules précurseurs et de modification de la densité cellulaire et de morphologie dans différentes régions cytoarchitecturales dans le SNC adulte 1-5. Dans un cerveau adulte en bonne santé, les microglies sont petites, ramifiées ou polaires cellules avec, des processus dynamiques fines. Contrairement aux périphériques macrophages morphologie, la microglie démontrent un repos, à profil bas phénotype dans les cerveaux sains qui peuvent apparaître comme l&#39;inactivité cellulaire, mais des études in vivo d&#39;imagerie montrent que les processus microgliales s&#39;étendent dynamiquement et se rétractent pour surveiller leur microenvironnement dans une manière qui rappelle &quot; l&#39;échantillonnage et l&#39;arpentage &quot;6,7. Les microglies sont fortement et de manière différentielle en réponse à des modifications environnementales et physiopathologiques dans le cerveau, de commutationd&#39;un surveillant à un pays considéré comme leur repos et activées couramment étatiques effectrices, respectivement. Ce commutateur dans l&#39;activation peut être médiée par l&#39;engagement des récepteurs membranaires reconnaissance de formes (PRR), tels que les récepteurs Toll-like (TLR), qui répondent aux motifs moléculaires associés à des pathogènes (PAMP), les lipoprotéines à savoir bactériennes et virales dérivés , des acides nucléiques, des hydrates de carbone et 8-11. En plus de PAMP, PRR ont également été montré pour induire l&#39;activation des microglies contre, des molécules stériles non pathogènes connus comme des modèles moléculaires danger / dommages-associé (atténue), qui représentent une perturbation dans l&#39;homéostasie du système nerveux central, tels que les dommages cellulaires 12-16. Une fois engagé, PRR initier une intracellulaire cascade qui se traduit par des changements dans la morphologie des cellules et l&#39;expression des gènes de la microglie de signalisation, plus précisément, les microglies activées adapter un phénotype de amoeboid-like, la translocation de la p65 de NF-kB sous-unité (p65) dans le noyau de la cellule, et réguler à la hausse l&#39; production et la sécrétion de cytokines pro-inflammatoires, tels que facteur de nécrose tumorale α (TNF-α), l&#39;interleukine-1 β (IL-1β), et aussi d&#39;espèces réactives de l&#39;oxygène (ROS) de 16 à 24. Bien intégré dans la réponse immunitaire innée du système nerveux central, ces molécules sécrétées ont également été trouvés à augmenter le stress oxydatif neuronale, induisant de ce fait et en exacerbant la neurodégénérescence dans les états malades telles que la maladie de Parkinson et la maladie d&#39;Alzheimer 25-29. Cependant, les mécanismes d&#39;activation de la microglie dans les états pathologiques ne sont pas complètement compris. Par conséquent, l&#39;isolement de la microglie est un puissant outil d&#39;enquête dans ces processus biologiques, comme beaucoup de caractéristiques in vivo de l&#39;activation de la microglie peut être récapitulé dans la culture. Plusieurs méthodes sont disponibles pour isoler les microglies, y compris l&#39;isolement par un gradient de Percoll après digestion enzymatique de tissu du SNC 30,31. Cependant, une digestion enzymatique peut modifierl&#39;immunophénotype des cellules en réduisant la surface cellulaire antigène expression 32, et les résultats dans le rendement de la cellule par animal inférieur que le procédé décrit ici. Plus précisément, nous présentons un rendement de microglies moyenne par chiot cortex de 7,5 x 10 5 cellules, alors que précédemment rapporté méthodes de l&#39;ensemble du système nerveux central d&#39;isolement par un rendement de gradient de Percoll 3-5 x 10 5 cellules 30,33,34. La présente procédure contourne l&#39;utilisation d&#39;enzymes de digestion en isolant la microglie en fonction de leurs propriétés de faible adhérence, préservant ainsi l&#39;immunophénotype des microglies et la fonctionnalité. Dans la présente étude, nous décrivons l&#39;isolement des cellules microgliales de cultures gliales mixtes issus de hétérozygotes CX3CR1-GFP (CX3CR1-GFP + / -), néonatale et C57BL / 6 cortex de souris par agitation mécanique sur un agitateur rotatif, une extension de déjà Procédé publiée 24,35. Nous utilisons la souche de souris ancien pour une visualisation facile des microglies, commeCes souris expriment la GFP sous le contrôle du locus endogène CX3CR1 - un promoteur spécifique des monocytes de 36 à 38. Cette méthode produit des cultures microgliales très pur avec un immunophénotype conservés ex vivo comme démontré par des changements morphologiques, la translocation nucléaire de p65, ainsi que la sécrétion de TNF-α en cas de contestation par le lipopolysaccharide bactérien (LPS) ou Pam 3 CSK 4, TLR4 et TLR1 / 2 agonistes , respectivement.

<table border="0" cellpadding="0" cellspacing="0" width="680">
	<colgroup>
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="160">
			<td height="160" style="height:160px;width:233px;">Glucose</td>
			<td style="width:149px;">Sigma</td>
			<td style="width:155px;">G8270</td>
			<td style="width:143px;">Make 20% Stock Solution with MilliQ water; filter sterilize; store at 4 &#176;C; shelf life: 3-6 months. Used to make MCM and MGM.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">Sodium pyruvate 100 mM (100x)</td>
			<td style="width:149px;">Hyclone</td>
			<td style="width:155px;">SH30239.01</td>
			<td style="width:143px;">Store at 4 &#176;C. Used to make MCM and MGM.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Penicillin/Streptomycin 10,000 units/ml (100x)</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:155px;">15140-122</td>
			<td style="width:143px;">Store at -20 &#176;C. Used to make MCM, MGM, MM, and DM.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">L-glutamine 200 mM (100x)</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:155px;">25030-081</td>
			<td style="width:143px;">Store at -20 &#176;C. Used to make MCM and MGM.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Fetal Bovine Serum (Defined)</td>
			<td style="width:149px;">Hyclone</td>
			<td style="width:155px;">SH30070.03</td>
			<td style="width:143px;">Filter sterilize; store at -20 &#176;C. Used to make MCM and MGM.</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:233px;">Minimum Essential Medium Earle&#39;s (MEM)</td>
			<td style="width:149px;">Cellgro</td>
			<td style="width:155px;">15-010-CV</td>
			<td style="width:143px;">Without L-glutamine. Contains Earle&#39;s salts. Used to make MCM, MGM, and DM.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Horse Serum</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:155px;">16050</td>
			<td style="width:143px;">Filter sterilize; store at -20 &#176;C. Used to make MCM and MGM.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Hanks&#39; Balanced Salt Solution (HBSS)</td>
			<td style="width:149px;">Cellgro</td>
			<td style="width:155px;">21-021-CV</td>
			<td style="width:143px;">Without calcium and magnesium. Store at 4 &#176;C. Used to make MM.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">HEPES 1 M</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:155px;">15630-031</td>
			<td style="width:143px;">Store at 4 &#176;C. Used to make MM.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">T-75 Flask</td>
			<td style="width:149px;">Corning</td>
			<td style="width:155px;">430641</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">4&#39;,6-Diamidino-2-Phenylindole, dilactate (DAPI)</td>
			<td style="width:149px;">Invitrogen</td>
			<td style="width:155px;">D3571</td>
			<td style="width:143px;">Used to stain cell nucleus.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">Rabbit anti-Iba1</td>
			<td style="width:149px;">Wako</td>
			<td style="width:155px;">019-19741</td>
			<td style="width:143px;">Used at 1/750 dilution for ICC staining of Iba1.&#160;</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">Rabbit anti-NF&#954;B (p65)</td>
			<td style="width:149px;">Abcam</td>
			<td style="width:155px;">7970</td>
			<td style="width:143px;">Used at 1/1250 dilution for ICC staining for p65.</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:233px;">Alexafluor 594 Goat anti-Rabbit IgG (H+L)</td>
			<td style="width:149px;">Invitrogen</td>
			<td style="width:155px;">A11012</td>
			<td style="width:143px;">Used at 1/1000 dilution for visualization of antigen:antibody complex in ICC.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">10 ml Disposable Serological Pipet</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:155px;">13-678-11E</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">50 ml Disposable Centrifuge Tube</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:155px;">05-539-8</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">15 ml Disposible Centrifuge Tube</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:155px;">05-539-12</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Sterile Polystyrene Petri Dish</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:155px;">875713</td>
			<td style="width:143px;">100 mm x 15 mm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Scissor: Straight Metzembaum (scissor #1)</td>
			<td style="width:149px;">Roboz Surgical</td>
			<td style="width:155px;">RS-6010</td>
			<td style="width:143px;">1; 5 inch; used for removing head</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Scissor: Vannas 
			(scissor #2)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">15000-08</td>
			<td style="width:143px;">1; non-angled; 2.5mm cutting edge; used to open scalp</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Scissor: Student Vannas (scissor #3)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">91501-09</td>
			<td style="width:143px;">1; curved; used to mince brain tissue</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:233px;">Forcep: Dumont #7 
			(forcep #1)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">91197-00</td>
			<td style="width:143px;">2; used to secure nose and remove cortices&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Forcep: Dumont #2 
			(forcep #2)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">11223-20</td>
			<td style="width:143px;">1; used to remove scalp</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Forcep: Dumont #3 
			(forcep #3)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">11231-30</td>
			<td style="width:143px;">1; used to remove skull</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Forcep: Dumont #5a 
			(forcep #4)</td>
			<td style="width:149px;">Fine Science Tools</td>
			<td style="width:155px;">11253-21</td>
			<td style="width:143px;">1; used to remove meninges</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Table of specific equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">Name of Equipment</td>
			<td style="width:149px;">Name of Company</td>
			<td style="width:155px;">Catalogue Number</td>
			<td style="width:143px;">Comments</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">Zoom Stereo Dissection Microscope&#160;</td>
			<td style="width:149px;">Olympus</td>
			<td style="width:155px;">SZ4060</td>
			<td style="width:143px;">Microscope is placed inside Laminar-Horizontal Flow Cabinet</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Laminar-Horizontal Flow Cabinet</td>
			<td style="width:149px;">Nuaire</td>
			<td style="width:155px;">NU-201-330</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Biological Safety Cabinet</td>
			<td style="width:149px;">Labconco</td>
			<td style="width:155px;">3440001</td>
			<td style="width:143px;">Class II</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:233px;">Water-Jacketed CO2 Incubator</td>
			<td style="width:149px;">VWR</td>
			<td>97025-836</td>
			<td style="width:143px;">Set to 37 &#176;C, 5% CO2</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Swing-out buckets</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>75006441</td>
			<td style="width:143px;">To be used with Swing-out rotor</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Swing-out Rotor</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>75006445</td>
			<td style="width:143px;">Max Radius: 19.2 (cm)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Sorvall Legend RT+ Centrifuge 
			(clinical centrifuge)</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>75-004-377</td>
			<td style="width:143px;">With swing-out rotor</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:233px;">AccuSpin Micro 17 microcentrifuge 
			(tabletop microcentrifuge)</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>S98645</td>
			<td style="width:143px;">With microliter rotor (24 x 1.5/2.0 ml; 
			Cat #: 75003524)&#160;&#160;</td>
		</tr>
	</tbody>
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isolation of cortical microglia with preserved immunophenotype and functionality from murine neonates

Isolation de la microglie du tissu du SNC est un puissant outil d&#39;enquête utilisé pour étudier la biologie de la microglie ex vivo. La présente méthode décrit en détail une procédure d&#39;isolement de la microglie à partir de cortex de souris néonatales par agitation mécanique avec un agitateur rotatif. Cet isolement de la microglie méthode donne de la microglie corticales très pures qui présentent des caractéristiques morphologiques et fonctionnelles indicatifs de la microglie de repos dans des conditions normales non pathologiques in vivo. Cette procédure préserve aussi l&#39;immunophénotype de la microglie et la fonctionnalité biochimique tel que démontré par l&#39;induction de changements morphologiques, la translocation nucléaire de la sous-unité p65 de NF-kB (p65), et la sécrétion de la cytokine marque de pro-inflammatoire, facteur de nécrose tumorale α (TNF-α ), sur le lipopolysaccharide (LPS) et Pam 3 CSK 4 (Pam) défis. Par conséquent, la procédure d&#39;isolement présente préserve l&#39;immunophénotype de repos et deux actimicroglie vée, en fournissant une méthode expérimentale d&#39;enquêter sur la microglie biologie dans des conditions ex vivo.

Le maintien de l&#39;immunophénotype et la fonctionnalité de la microglie ex vivo lors de l&#39;isolement est essentiel d&#39;être en mesure d&#39;utiliser ces cellules comme un modèle d&#39;enquête pour la biologie de la microglie. Afin de démontrer la préservation de la microglie immunofunctionality succès en utilisant la présente méthode, on a isolé à partir de la microglie corticales nouveau-nés (P3 CX3CR1-GFP + / - et des souris C57BL / 6) et des cultures traitées soit avec du LPS ...

Watch this Scientific Journal Video about Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates at JoVE.com

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

Une des clés de réussite de l&#39;enquête de la biologie de la microglie est la préservation de la microglie immunofunction ex vivo lors de l&#39;isolement de tissu du SNC. Isoler la microglie par les résultats serrant rotatifs dans des cultures de cellules de grande pureté et immunofonctionnel évaluée par imagerie de fluorescence, immunocytochimie, et ELISA activation de la microglie suivante avec le lipopolysaccharide stimuli pro-inflammatoires (LPS) et Pam 3 CSK 4 (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-cortical-microglia-with-preserved-immunophenotype

Research

JoVE Journal

Immunology and Infection

15.6K vues.  Georgetown University Medical Center.  Une des clés de réussite de l'enquête de la biologie de la microglie est la préservation de la microglie immunofunction ex vivo lors de l'isolement de tissu du SNC. Isoler la microglie par les résultats serrant rotatifs dans des cultures de cellules de grande pureté et immunofonctionnel évaluée par imagerie de fluorescence, immunocytochimie, et ELISA activation de la microglie suivante avec le lipopolysaccharide stimuli pro-inflammatoires (LPS) e...

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

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 ...

Recherche

Immunologie et infection

Isolation, Processing and Analysis of Murine Gingival Cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.

This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.

Isolation, traitement et analyse des cellules gingivales murins

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

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 Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Isolement de cellules murines ganglions lymphatiques stromales

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

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 and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

Isolement et activation de murins Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during physiological and pathological states. The isolation of EVs continues to be a major challenge in this field, due to limitations intrinsic to each technique. The differential ultracentrifugation with density gradient centrifugation method is a commonly used approach and is considered to be the gold standard procedure for EV isolation. However, this procedure is time-consuming, labor-intensive, and generally results in low scalability, which may not be suitable for small-volume samples such as bronchoalveolar lavage fluid. We demonstrate that an ultrafiltration centrifugation isolation method is simple and time- and labor-efficient yet provides a high recovery yield and purity. We propose that this isolation method could be an alternative approach that is suitable for EV isolation, particularly for small-volume biological specimens.

Here, we describe two extracellular vesicle isolation protocols, ultrafiltration centrifugation and ultracentrifugation with density gradient centrifugation, to isolate extracellular vesicles from murine bronchoalveolar lavage fluid samples. The extracellular vesicles derived from murine bronchoalveolar lavage fluid by both methods are quantified and characterized.

Isolement des vésicules extracellulaires du murin lavage broncho-alvéolaire fluide à l’aide d’une Technique de Centrifugation de l’Ultrafiltration

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during ...

Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of massage on skeletal muscles, the molecular mechanisms behind are poorly understood. We have recently developed a simple device to apply local cyclical compression (LCC), which can generate intramuscular pressure waves with varying amplitudes. Using this device, we have demonstrated that LCC modulates inflammatory responses of macrophages in situ and alleviates immobilization-induced muscle atrophy. Here, we describe protocols for the optimization and application of LCC as a massage-like intervention against immobilization-induced inflammation and atrophy of skeletal muscles of mouse hindlimbs. The protocol that we have developed can be useful for investigating the mechanism underlying beneficial effects of physical exercise and massage. Our experimental system provides a prototype of the analytical approach to elucidate the mechanical regulation of muscle homeostasis, although further development needs to be made for more comprehensive studies.

Here we describe the protocols for applying defined mechanical loads to mouse calves and for monitoring the concomitant intramuscular pressure changes. The experimental systems that we have developed can be useful for investigating the mechanism behind the beneficial effects of physical exercise and massage.

Application de perturbations de massage cohérentes sur les veaux de souris et surveillance des changements de pression intramusculaire qui en résultent

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of ...

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn&#8217;s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

Isolement des cellules mononucléaires Lamina Propria du colon murine à l'aide de la collagène E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...

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 ...