Nous tenons à remercier les membres du laboratoire Tsirka pour leurs conseils et leurs commentaires utiles. Ce travail a été soutenu par R01NS42168 à définir, 12PRE12060489 de RB, un NSF-3MT IGERT et une thèse bourse Turner à LT, et NSF-3MT IGERT à JCN.

1. Dissection (jour 0) <ol><li> Induire une anesthésie à P0-2 souriceaux d&#39;hypothermie. Essuyez les têtes des chiots avec un Kimwipe trempé dans l&#39;éthanol à 70% pour désinfecter les tissus. </li><li> Enlevez les têtes avec une paire de ciseaux, avec 4 petits par plaque de culture de 10 cm. Placez les têtes dans une boîte de Pétri contenant le tampon glacée Hank. </li><li> Ancrer les têtes avec une pince courbe à travers les orbites et retirer délicatement la peau qui recouvre le crâne avec microforceps droites. </li><li> Retirez les os du crâne avec microforceps droites, en utilisant attention à ne pas percer ou endommager le cortex. Le moyen le plus efficace est de commencer le déménagement de départ près du cervelet, qui est situé à la base de la tête, car cette région sera jeté. </li><li> Retirez le cerveau avec une petite spatule et placer les quatre (4) des cerveaux dans une boîte de Pétri frais contenant le tampon glacée Hank. </li><li> Toutes les étapes de ce point sur utilisent microforceps et sont réalisées sous une dissemicroscope ction. À partir de la face ventrale du cerveau, ancrer le tissu en tenant le cervelet et faire deux petites incisions de chaque côté du mésencéphale. Veillez à ne pas couper tout le chemin à travers le tissu, comme les cortex couvrent le mésencéphale dans cette région. </li><li> Doucement taquiner le mésencéphale et le cervelet en une seule pièce les deux corticales. Les deux cortex devraient former une forme concave. </li><li> Séparer les deux cortex et orienter un seul cortex avec la face interne en vue de poursuivre la dissection. L&#39;hippocampe peut être difficile à observer, mais il est situé en face du bulbe olfactif qui apparaît comme un petit nodule sur l&#39;extrémité pointue du cortex. </li><li> Retirez l&#39;hippocampe, qui a une forme de croissant. Retournez le cortex plus à voir la face dorsale. </li><li> Utilisation du bulbe olfactif comme point de départ, supprimer toutes les méninges et le bulbe olfactif lui-même à partir du cortex. </li><li> Les cortex disséqués devront être placés dans des tubes coniques de 15 ml avec 14 ml de colLa saline tamponnée d Hank sur la glace. </li></ol> 2. Culture cellulaire (jour 0) <ol><li> Pré-manteau 10 boîtes de culture de tissus cm avec 5 ug / ml de poly-D-lysine (PDL) dilué dans de l&#39;eau stérilisée à l&#39;autoclave pendant 3 heures à 37 ° C. </li><li> Aspirer PDL, et des plaques de lavage une fois avec de l&#39;eau autoclave. Plaque à sec sous les UV dans la hotte de culture tissulaire pendant 20 min. Cette étape doit être effectuée juste avant le processus de dissection. </li><li> Aspirer le tampon de Hank à partir de tubes contenant les cortex de 4 cerveaux chacun, appliquer 4 ml de solution de trypsine / EDTA 1x, triturer tissu avec p1000 pointe, et placer des tubes à 37 ° C incubateur pendant 15 min. </li><li> Ajouter 4 ml de milieu microgliales complets par tube pour arrêter la digestion enzymatique, mélanger et ralentit contenu à un régime 1.5K pendant 5 min. </li><li> Aspirer le surnageant et répéter lavage avec 4 ml de milieu complet. Remettre les cellules dans 10 ml de milieu microgliales complets (DMEM avec 10% de FBS, 1% de pyruvate de sodium, 0,08% gentamicine) et filtrer à travers un 40 micron tamis cellulaire maille. </li><li> Plaque à une densité de 8 cortex par 10 ml dans une plaque de 10 cm de culture de tissu et placer dans un incubateur de culture tissulaire à 37 ° C et 5% de CO 2 (voir protocole Microglia adultes). </li></ol> (Jour 3) <ol><li> Changer médias dans tous les plats de culture de cellules microgliales complets avec les médias. </li></ol> (Jour 10) <ol><li> Ajouter 400 microlitres de 60 mm de lidocaïne dans HBSS (pour détacher la microglie) dans le milieu des plaques de culture tissulaire de 10 cm et incuber à température ambiante (TA) pendant 10-15 min. </li><li> Recueillir des médias / suspension cellulaire de la plaque et laver la plaque une fois avec le tampon de Hank. Recueillir le tampon de lavage restant à recouvrer la microglie. </li><li> Ajouter EDTA 5 mM (pH 8,0) à la suspension de cellules à une concentration finale de 50 pM ou à une dilution de 1/100. </li><li> Isoler suspension cellulaire à 1000 x g (1500 rpm) pendant 5 min et remettre en suspension dans 1 ml de DMEM avec 1% de FBS. </li><li> Compter le nombre de cellules viables (comme par Mi adultecroglia 3.1/3.2) et les cellules divisée en plaques de culture de tissus à la densité expérimentale souhaitée. Environ 1x10 6 cellules microgliales sont récoltées à partir d&#39;une plaque de 10 cm contenant les cortex de 4 cerveaux. Par immunofluorescence, une densité de 2,5.10 4 cellules par lamelle 18 mm est recommandé. </li><li> Attendre au moins 24 heures pour les cellules microgliales pour revenir pleinement à leur état de repos ramifié avant de l&#39;utiliser. </li></ol> Protocole texte: (Adult microglie) 1. Collection de tissus <ol><li> des plaques de culture de tissus de manteau de poly-D-lysine (PDL) pendant 3 heures à 37 ° C ou une nuit à 4 ° C. Laver les plaques une fois avec de l&#39;eau immédiatement autoclave avant utilisation. </li><li> Euthanasier souris par asphyxie au CO2 et de s&#39;assurer que l&#39;euthanasie est terminée. Nettoyer la zone de la moelle épinière avec 70% d&#39;éthanol. </li><li> En utilisant une paire de ciseaux couper la peau sur le dessus de la moelle épinière, puis passez à couper la moelle épinière à partir région T1 à T12. Couperle muscle restant hors des côtés de la moelle épinière. </li><li> Lentement couper les vertèbres en utilisant microciseaux que vous maintenez doucement la moelle épinière avec vos doigts. Faites attention à ne pas percer la moelle épinière que le tissu est très doux. Extraire lentement la moelle épinière à l&#39;aide microforceps. </li><li> Immerger la moelle épinière dans une boîte de Pétri contenant HBSS glacée. L&#39;utilisation d&#39;un microscope optique retirer toutes méninges visibles à l&#39;aide de microforceps </li><li> Couper la moelle épinière en segments transversaux suffisamment petites pour générer plusieurs fragments. L&#39;épaisseur des fragments ne doit pas être supérieure à 2 mm, afin de faciliter la digestion efficace. Transférer les morceaux de la moelle épinière dans un tube conique de 15 ml contenant 1 ml de HBSS glacée. Garder le tube sur la glace jusqu&#39;à l&#39;étape de digestion. </li></ol> 2. La digestion des tissus <ol><li> Aspirer HBSS de 15 ml tube conique contenant du tissu de la moelle épinière. Veillez à ne pas aspirer n&#39;importe quel morceau de tissu. </li><li> Ajouter 1 ml de trypsine (2.5 g / L irradié trypsine porcine et 0,2 g / L EDTA dans HBSS) et incuber à 37 ° C pendant 30 min. </li><li> Pour arrêter la digestion retirer la trypsine et ajouter 3 ml de milieu microglie primaire qui contient du sérum pour arrêter la digestion enzymatique. </li><li> Pipet haut et bas pour déloger le tissu. Filtrer la suspension de cellules à l&#39;aide d&#39;un tamis cellulaire de 40 um. </li></ol> 3. Comptage des cellules et placage <ol><li> Mélanger volume égal de suspension de cellules et une solution de DAPI. </li><li> Compter les cellules en utilisant un hématimètre. </li><li> Plaque à une densité comprise entre 5 7x10 5 cellules / ml dans PDL revêtus 35 x 10 mm plats. Placage à une densité inférieure empêché la croissance des cellules, même lorsque les cellules ont été cultivées en plaques de 12 puits qui ont une zone plus petite par rapport à 35 x 10 mm plats. </li></ol>

<ol>
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Les auteurs n&#39;ont rien à révéler.

Microglie moduler CNS fonctionnement normal ainsi que les réponses inflammatoires à diverses pathologies. Fonctionnel remodelage synaptique par la microglie a été impliquée dans le maintien de l&#39;homéostasie du cerveau normal 15. Au cours de la cascade neurogène ils participent à la clairance des cellules progénitrices neurales du gyrus denté de l&#39;hippocampe 4, 16. Par conséquent, il est nécessaire de développer un système de culture dans lequel pour étudier la microglie néon...

Microglies sont les cellules immunitaires résidentes du SNC, ressemblant plus étroitement macrophages périphériques dans la structure et la fonction 1. Il a été récemment démontré que les cellules microgliales postnatale proviennent de progéniteurs myéloïdes primitives et sont générés avant le huitième jour de l&#39;embryogenèse, bouleversant la notion précédente qui progéniteurs hématopoïétiques postnatales sont la source de la microglie dans le cerveau adulte 2. Ils jouent un rôle clé dans plusieurs maladies neurologiques et peuvent réagir rapidement à une infection ou une blessure en libérant des cytokines pro-inflammatoires ou anti-inflammatoire 3. Ainsi microglie englober une unité autonome dans le système nerveux central qui peuvent être manipulés à affecter la progression de la maladie. Développer des méthodes robustes et reproductibles pour isoler et cultiver les cellules microgliales néonatale ou adulte est important pour de futures études. Les microglies sont connus pour être des acteurs essentiels dans un certain nombre de pathologies cérébrales. Plus récemment, roles sont en train d&#39;émerger pour les cellules dans le développement normal du cerveau et de la fonction que les cellules microgliales phagocytent excès de neurones progéniteurs du gyrus denté de l&#39;hippocampe 1, 4. Microglie peut aussi moduler plusieurs conditions neurologiques qui affectent la moelle épinière, comme MS, les douleurs neuropathiques et les blessures de la moelle épinière 5-7. Spinal microglies de la moelle réagissent différemment par rapport à la microglie du cerveau en réponse à des signaux d&#39;activation 8, 9, probablement en raison de différences dans l&#39;environnement local. Il est donc important de mettre en place un système in vitro approprié à la culture et à étudier les microglies de la moelle épinière. Microglie néonatale produisent beaucoup plus de l&#39;oxyde de cytokines pro-inflammatoires nitrique par rapport aux cellules adultes après stimulation in vitro avec l&#39;IFN-γ ou TNF-a 10,11 soulignant la nécessité d&#39;utiliser des cellules adultes pour étudier la microglie dans le cadre de certaines maladies plus loin. Le protocole que nous utilisons en laboratoire pour culture microglie néonatale est une variante de méthodes récentes qui utilisent tremblement de cultures mixtes gliales dans un effort pour supprimer les microglies de la surface du flacon de culture cellulaire 12. Nous décrivons également une méthode de culture microglies de la moelle épinière de souris adulte sur base d&#39;un protocole d&#39;abord décrit par Yip, et al 13. Cette méthode fournit un moyen plus rapide de cellules adultes de la culture par rapport à d&#39;autres protocoles disponibles 14. La préparation obtenue est de 70% microglie, le pourcentage restant se compose d&#39;astrocytes. Bien que la pureté de notre culture est plus faible par rapport à d&#39;autres méthodes publiées 13, ce système de culture est utile pour explorer la microglie dans la réponse de la culture à divers stimuli d&#39;activation ainsi que pour l&#39;étude des maladies qui affectent principalement la moelle épinière et dans lequel une forte réponse inflammatoire est une caractéristique principale. Tous les protocoles décrits ont été approuvés par la Stony Brook University IACUC.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		 </tr>
		 <tr>
			<td>EDTA, 5mM</td>
			<td>Invitrogen</td>
			<td>15567-028</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Lidocaine, 60mM</td>
			<td>Sigma</td>
			<td>L-5647</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trypan Blue</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trypsin/EDTA</td>
			<td>Cellgro</td>
			<td>25-052-CI</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM, 1X</td>
			<td>Cellgro</td>
			<td>10-017</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium Pyruvate</td>
			<td>Cellgro</td>
			<td>25-000-Cl</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Gentamycin Sulfate</td>
			<td>Biowittaker</td>
			<td>17-518Z</td>
			<td></td>
		 </tr>
		 <tr>
			<td>35 x 10mm tissue culture dish</td>
			<td>Falcon</td>
			<td>353001</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Poly-D-Lysine, 100 &mu;g/ml</td>
			<td></td>
			<td></td>
			<td>Dilute 1:20</td>
		 </tr>
		 <tr>
			<td>HBSS, 1X</td>
			<td>Cellgro</td>
			<td>20-023-CV</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

culturing microglia from the neonatal and adult central nervous system

Les microglies sont les cellules de type macrophage résident du système nerveux central (SNC) et, à ce titre, ont un rôle d&#39;une importance cruciale dans les processus physiologiques et pathologiques tels que SNC maturation dans le développement, la sclérose en plaques et les lésions de la moelle épinière. Microglie peut être activé et recruté à l&#39;action par une blessure ou une stimulation neuronale, tels que des lésions axonales vu dans MS ou un traumatisme cérébral ischémique résultant d&#39;une attaque. Ces membres immunocompétentes du SNC sont également pensé à avoir un rôle dans la plasticité synaptique dans des conditions non pathologiques. Nous utilisons des protocoles pour la microglie de culture des tissus néonatales et adultes qui visent à maximiser le nombre de cellules viables tout en minimisant les facteurs de confusion, tels que la présence d&#39;autres types de cellules du système nerveux central et les débris de culture cellulaire. Nous utilisons des composants du système nerveux central, grandes et facilement discernable (par exemple cortex, les segments de la moelle épinière), ce qui rend l&#39;ensemble du processus réalisable et reproductible. L&#39;utilisation de la cellule adultes est une alternative appropriée à l&#39;utilisation de la microglie du cerveau néonatal, comme beaucoup de pathologies étudiées touchent principalement la moelle épinière postnatal. Ces systèmes de culture sont également utiles pour tester directement l&#39;effet des composés qui peuvent inhiber ou promouvoir activation de la microglie. Depuis activation de la microglie peut façonner les résultats de la maladie dans le SNC adulte, il existe un besoin pour des systèmes in vitro dans lequel la microglie néonatale et adulte peut être cultivée et étudiée.

<html> Un exemple de cellules de la microglie au repos et activées est illustré à la figure 1. La microglie ont été visualisée 24 heures après placage (figure 1a) et présentent ramifié morphologie (au repos). L&#39;exposition au réactif d&#39;amorçage, le lipopolysaccharide (LPS) bactériens résultats des changements dans la morphologie des microglies que les cellules sont activées (figure 1b). Un exemple de comptage des cellules...

Watch this Scientific Journal Video about Culturing Microglia from the Neonatal and Adult Central Nervous System at JoVE.com

Culturing Microglia from the Neonatal and Adult Central Nervous System

Nous présentons des méthodes pour l&#39;isolement / culture efficace et rapide de la microglie viable du cortex cérébral néonatal et la moelle épinière adulte. La dissection et le placage de la microglie corticale peuvent être accomplies dans les 90 minutes, avec la récolte des microglies subséquente aura lieu ~ 10 jours suivant la dissection initiale.

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 ...

culturing-microglia-from-the-neonatal-and-adult-central-nervous-system

Research

JoVE Journal

Immunology and Infection

27.6K vues.  Stony Brook University. Nous présentons des méthodes pour l'isolement / culture efficace et rapide de la microglie viable du cortex cérébral néonatal et la moelle épinière adulte. La dissection et le placage de la microglie corticale peuvent être accomplies dans les 90 minutes, avec la récolte des microglies subséquente aura lieu ~ 10 jours suivant la dissection initiale.

Vidéo: Culturing Microglia from the Neonatal and Adult Central Nervous System

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

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

Purification et visualisation de lipopolysaccharide de bactéries à Gram négatif par Hot-phénol aqueux d&#39;extraction

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

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 ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Développement d&#39;un système de virus de l&#39;hépatite B Reporter pour surveiller les premiers stades du cycle de réplication

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

This protocol provides an analysis of the macrophage subpopulations in the adult mouse central nervous system by flow cytometry and is helpful for the study of multiple markers expressed by these cells.

Analyse de la microglie et des macrophages dérivés de monocytes du système nerveux central par cytométrie de flux

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

Isoler des Lymphocytes du système immunitaire Intestinal souris petit

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

A Positioning Device for the Placement of Mice During Intranasal siRNA Delivery to the Central Nervous System

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the central nervous system (CNS). Recent studies demonstrate the use of a peptide, RVG9R, incorporating the minimal receptor-binding domain of the Rabies virus glycoprotein, in eliciting the delivery of siRNA into neurons in the brain. In this protocol, the peptide-siRNA formulation is delivered intranasally with a pipette in the dominant hand, while the anesthetized mouse is restrained by the scruff with the nondominant hand in a "head down-and-forward position" to avoid drainage into the lung and stomach upon inhalation. This precise gripping of mice can be learned but is not easy and requires practice and skill to result in effective CNS uptake. Furthermore, the process is long-drawn, requiring about 45 min for the administration of a total volume of ~20-30 &#181;L of solution in 1-2 &#181;L droplet volumes per inhalation, with 3-4 min rest periods between each inhalation. The objective of this study is to disclose a mouse positioning device that enables the appropriate placement of mice for efficient IN administration of the peptide-siRNA formulation. Multiple features are incorporated into the design of the device, such as four or eight positioning chairs with adjustable height and tilt to restrain anesthetized mice in the head down-and-forward position, enabling easy visualization of the mice's nares and a built-in heating pad to maintain the mice's body temperatures during the procedure. Importantly, the ability to treat four or eight mice simultaneously with RVG9R-siRNA complexes in this manner enables studies on a much quicker time scale, for the testing of an IN therapeutic siRNA approach. In conclusion, this device allows for appropriate and controlled mouse head positioning for IN application of RVG9R-siRNA and other therapeutic molecules, such as nanoparticles or antibodies, for CNS delivery.

Here, we present a protocol using a mouse positioning device that enables the appropriate placement of mice for the intranasal administration of a brain-targeting peptide-siRNA formulation enabling effective gene silencing in the central nervous system.

Un dispositif de positionnement pour le placement des souris pendant la livraison de siRNA intranasal au système nerveux central

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the ...

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

Analyse cytométrique du flux de l’infiltration de lymphocytes dans le système nerveux central pendant l’encéphalomyélite autoimmune expérimentale

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...

Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier between the periphery and the CNS. The CNS is an immunologically specialized site, and in steady state conditions, immune privilege is most evident in the CNS parenchyma. In contrast, the meninges harbor a diverse array of resident cells, including innate and adaptive immune cells. During inflammatory conditions triggered by CNS injury, autoimmunity, infection, or even neurodegeneration, peripherally derived immune cells may enter the parenchyma and take up residence within the meninges. These cells are thought to perform both beneficial and detrimental actions during CNS disease pathogenesis. Despite this knowledge, the meninges are often overlooked when analyzing the CNS compartment, because conventional CNS tissue extraction methods omit the meningeal layers. This protocol presents two distinct methods for the rapid isolation of murine CNS tissues (i.e., brain, spinal cord, and meninges) that are suitable for downstream analysis via single-cell techniques, immunohistochemistry, and in situ hybridization methods. The described methods provide a comprehensive analysis of CNS tissues, ideal for assessing the phenotype, function, and localization of cells occupying the CNS compartment under homeostatic conditions and during disease pathogenesis.

This paper presents two optimized protocols for examining resident and peripherally derived immune cells within the central nervous system, including the brain, spinal cord, and meninges. Each of these protocols helps to ascertain the function and composition of the cells occupying these compartments under steady state and inflammatory conditions.

Isolement des tissus du système nerveux central et des méninges associées pour l’analyse en aval des cellules immunitaires

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier ...

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (&#62;10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells&#160;(DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.

Protocole économique et efficace pour isoler et cultiver des cellules dendritiques dérivées de la moelle osseuse à partir de souris

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method ...