Vorremmo ringraziare i membri del Tsirka laboratorio per i loro consigli e utili commenti. Questo lavoro è stato sostenuto da R01NS42168 a SET, 12PRE12060489 a RB, un NSF-3MT IGERT e un Turner Tesi borsa di studio a LT, e NSF-3MT IGERT a JCN.

1. Dissection (giorno 0) <ol><li> Indurre l&#39;anestesia per p0-2 cuccioli di topo con ipotermia. Pulire le teste dei cuccioli con un Kimwipe imbevuto di etanolo al 70% per disinfettare il tessuto. </li><li> Togliere le teste con un paio di forbici, con 4 cuccioli per 10 cm piastra di coltura. Mettere le teste in una capsula di Petri contenente tampone di ghiaccio-freddo Hank. </li><li> Ancorare le testine utilizzando pinze curve attraverso le orbite e rimuovere con attenzione la pelle che ricopre il cranio con microforceps dritte. </li><li> Togliere le ossa craniche con microforceps rette, prestando attenzione a non forare o danneggiare la corteccia. Il modo più efficace è quello di iniziare la rimozione partire vicino il cervelletto, che si trova alla base della testa, come sarà scartata questa regione. </li><li> Togliere il cervello con una spatolina, e posizionare le (4) cervelli in una capsula di Petri contenente tampone fresco di ghiaccio freddo Hank. </li><li> Tutti i passi da questo punto in poi utilizzano microforceps e vengono eseguite sotto una diffuction microscopio. Partendo dal lato ventrale del cervello, ancorare il tessuto tenendo il cervelletto e fare due piccole incisioni ai lati del mesencefalo. Fare attenzione a non tagliare tutto il percorso attraverso il tessuto, come le cortecce coprono il mesencefalo in questa regione. </li><li> Stuzzicare delicatamente il mesencefalo e cervelletto in un pezzo da due cortecce. Le due cortecce dovrebbero formare una forma concava. </li><li> Separare le due cortecce e orientare una sola corteccia con il lato mediale per ulteriore dissezione. L&#39;ippocampo può essere difficile da osservare, ma si trova di fronte del bulbo olfattivo che appare come un piccolo nodulo sull&#39;estremità appuntita della corteccia. </li><li> Rimuovere l&#39;ippocampo, che ha una forma di mezzaluna. Capovolgere la corteccia sopra per vedere il lato dorsale. </li><li> Utilizzando il bulbo olfattivo come punto di partenza, rimuovere tutte le meningi e il bulbo olfattivo stesso dalla corteccia. </li><li> Le cortecce sezionati devono essere collocati in provette coniche da 15 ml con 14 ml di Colsoluzione fisiologica tamponata di d Hank su ghiaccio. </li></ol> 2. Colture Cellulari (giorno 0) <ol><li> Pre-coat 10 centimetri di tessuto piastre di coltura con 5 pg / ml di poli-D-lisina (PDL) diluito in acqua autoclavato per 3 ore a 37 ° C. </li><li> Aspirare Pdl, e piatti lavare una volta con acqua sterilizzata. Monodisco a secco sotto l&#39;UV nella cappa coltura tissutale per 20 min. Questa operazione deve essere eseguita appena prima del processo di dissezione. </li><li> Tampone di Hank aspirato dalle provette contenenti le cortecce da 4 cervelli ciascuno, applicare 4 ml di soluzione 1x tripsina / EDTA, triturare tessuto con p1000 punta, e posto i tubi nel 37 ° C incubatore per 15 min. </li><li> Aggiungere 4 ml di mezzi completi microgliali per tubo per fermare la digestione enzimatica, mescolare e centrifugare contenuto a 1.5K rpm per 5 min. </li><li> Aspirare il surnatante e ripetere lavata con 4 ml di mezzi completi. Risospendere le cellule in 10 ml di mezzi microgliali completi (DMEM con 10% FBS, 1% sodio piruvato, 0.08% gentamicina) e filtrare attraverso un 40 micron colino cellula maglia. </li><li> Piastra ad una densità di 8 cortecce per 10 ml in una piastra di coltura 10 cm tissutale e posto in una coltura tissutale incubatore a 37 ° C e 5% di CO 2 (Cfr. adulto protocollo Microglia). </li></ol> (Giorno 3) <ol><li> Modifica dei media in tutti i piatti di coltura di cellule microgliali con mezzi completi. </li></ol> (Giorno 10) <ol><li> Aggiungere 400 microlitri di 60 mm lidocaina in HBSS (per staccare microglia) in media di 10 cm di piastre di coltura di tessuto e incubare a temperatura ambiente (RT) per 10-15 min. </li><li> Raccogliere i media / sospensione di cellule dalla piastra, e lavare la piastra una volta con tampone di Hank. Raccogliere il tampone di lavaggio per recuperare rimanendo microglia. </li><li> Aggiungere 5 mM EDTA (pH 8,0) alla sospensione cellulare ad una concentrazione finale di 50 mM o ad una diluizione 1/100. </li><li> Spin down sospensione cellulare a 1.000 xg (1.500 rpm) per 5 min e risospendere in 1 ml di DMEM con 1% FBS. </li><li> Contare il numero di cellule vitali (come per Adulto Microglia 3.1/3.2) e celle divise in piastre di coltura di tessuti a densità sperimentale desiderato. Circa 1x10 6 cellule microgliali vengono raccolte da una piastra di 10 cm contenente le cortecce da 4 cervelli. Per immunofluorescenza, si raccomanda una densità di 2.5x10 4 cellule per 18 millimetri coprioggetto. </li><li> Lasciare almeno 24 ore per le cellule microgliali per tornare pienamente alla loro ramificata, stato di riposo prima dell&#39;uso. </li></ol> Protocollo Testo: (Adult microglia) 1. Collezione tessuti <ol><li> Cappotto tessuto piastre di coltura con poli-D-lisina (PDL) per 3 ore a 37 ° C o per una notte a 4 ° C. Lavare le piastre una volta con acqua autoclavata immediatamente prima dell&#39;uso. </li><li> Euthanize topo da CO 2 asfissia e di accertare che l&#39;eutanasia è completa. Pulire la zona del midollo spinale con il 70% di etanolo. </li><li> Usando un paio di forbici tagliare la pelle sulla parte superiore del midollo spinale; quindi procedere a tagliare il midollo spinale dalla regione T1 a T12. Tagliareil muscolo rimanenti fuori dei lati del midollo spinale. </li><li> Tagliare lentamente il vertebre utilizzando microscissors come si tiene delicatamente il midollo spinale con la punta delle dita. Siate attenti a non forare il midollo spinale come il tessuto è molto morbido. Lentamente estrarre il midollo spinale usando microforceps. </li><li> Immergere il midollo spinale in una capsula di Petri contenente HBSS ghiacciate. Utilizzando un microscopio ottico rimuovere tutti meningi visibili utilizzando microforceps </li><li> Tagliate il midollo spinale in segmenti trasversali sufficientemente piccole per generare diversi frammenti. Lo spessore dei frammenti non deve essere superiore a 2 mm, per facilitare la digestione efficiente. Trasferire i pezzi del midollo spinale di un tubo da 15 ml contenente 1 ml HBSS ghiacciate. Tenere il tubo in ghiaccio fino alla fase di digestione. </li></ol> 2. Digestione del Tissue <ol><li> Aspirare HBSS da 15 ml tubo conico contenente tessuto del midollo spinale. Fare attenzione a non aspirare qualsiasi pezzo di tessuto. </li><li> Aggiungere 1 ml di tripsina (2.5 g / L irradiati suina tripsina e in HBSS 0,2 g / L EDTA) e incubare a 37 ° C per 30 min. </li><li> Per fermare la digestione rimuovere la tripsina e aggiungere 3 ml di mezzo microglia primaria che contiene siero di arrestare la digestione enzimatica. </li><li> Pipettare su e giù per rimuovere il tessuto. Filtrare la sospensione cellulare utilizzando un colino cella 40 micron. </li></ol> 3. Conta delle cellule e placcatura <ol><li> Mescolare uguale volume di sospensione cellulare e la soluzione DAPI. </li><li> Contare le cellule utilizzando un emocitometro. </li><li> Piastra ad una densità compresa tra 5-7x10 5 cellule / ml nel PDL rivestite 35 x 10 piatti mm. Placcatura a una densità minore di prevenire la crescita cellulare anche quando le cellule sono state coltivate in piastre da 12 aventi un&#39;area più piccola rispetto a 35 x 10 piatti mm. </li></ol>

<ol>
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Microglia modulare CNS normale funzionamento così come le risposte infiammatorie a diverse patologie. Funzionale rimodellamento sinaptico da microglia è stata implicata nel mantenimento dell&#39;omeostasi normale del cervello 15. Durante la cascata neurogena partecipano alla liquidazione di cellule progenitrici neurali del giro dentato dell&#39;ippocampo 4, 16. Pertanto, è necessario sviluppare un sistema di coltura in cui studiare microglia neonatale e adulto, che coprirà la vasta fascia di sv...

Microglia sono le cellule immunitarie residenti del SNC, maggiormente somiglianti macrofagi periferici per struttura e funzione 1. E &#39;stato recentemente dimostrato che le cellule microgliali postnatali derivano da progenitori mieloidi primitivi e sono generati prima dell&#39;ottavo giorno di embriogenesi, rovesciando il concetto precedente che postnatali progenitori emopoietici sono la fonte della microglia nel cervello adulto 2. Essi giocano un ruolo chiave in diverse malattie neurologiche e possono rispondere rapidamente alle infezioni o lesioni attraverso il rilascio di citochine pro-infiammatorie e anti-infiammatori 3. Così microglia comprendono una unità autonoma nel SNC che possono essere manipolati per influenzare la progressione della malattia. Lo sviluppo di metodi robusti e riproducibili per isolare e coltivare le cellule microgliali neonatali o adulto è importante per gli studi futuri. Microglia sono noti per essere giocatori critici in un certo numero di patologie cerebrali. Più di recente, roles stanno emergendo per le cellule nel normale sviluppo del cervello e la funzione come microglia fagocitare eccesso di cellule progenitrici neurali del giro dentato dell&#39;ippocampo 1, 4. Microglia può anche modulare le diverse condizioni neurologiche che colpiscono il midollo spinale, come MS, dolore neuropatico, e le lesioni del midollo spinale 5-7. Microglia midollo spinale reagiscono in modo diverso rispetto al cervello microglia in risposta ai segnali di attivazione 8, 9, probabilmente a causa delle differenze nell&#39;ambiente locale. Quindi è importante stabilire un adeguato sistema in vitro per studiare la cultura e microglia midollo spinale. Microglia neonatali producono significativamente maggiore della citochina pro-infiammatoria ossido di azoto rispetto a cellule adulte dopo stimolazione in vitro con IFN-γ o TNF-a 10,11 un&#39;ulteriore evidenziando la necessità di utilizzare cellule adulte per studiare microglia nel contesto di alcune malattie. Il protocollo impieghiamo in laboratorio per culture microglia neonatale è una variazione di metodi recenti che utilizzano agitazione di colture gliali miste nel tentativo di rimuovere la microglia dalla superficie del pallone di coltura cellulare 12. Noi descriviamo anche un metodo per cultura microglia dal midollo spinale adulto topo basata su un protocollo prima descritto da Yip, et al 13. Questo metodo fornisce un modo più rapido per le cellule adulte della cultura rispetto ad altri protocolli disponibili 14. La preparazione risultante è del 70% microglia; la percentuale rimanente è costituita da astrociti. Sebbene la purezza della nostra cultura è più basso rispetto ad altri metodi pubblicati 13, questo sistema di coltura è utile per esplorare la microglia in coltura risposta a vari stimoli attivanti e anche per lo studio di malattie che colpiscono principalmente il midollo spinale e in cui una forte risposta infiammatoria è una caratteristica principale. Tutti i protocolli descritti sono stati approvati dalla 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

Microglia sono i residenti i macrofagi del sistema nervoso centrale (SNC) e, come tali, hanno ruoli criticamente importanti in processi fisiologici e patologici quali CNS maturazione in sviluppo, sclerosi multipla, e lesioni del midollo spinale. Microglia può essere attivato e reclutato per azione da lesioni o la stimolazione neuronale, come il danno assonale visto in MS o di un trauma cerebrale ischemico derivanti da ictus. Questi membri immunocompetenti del SNC sono anche pensato di avere un ruolo nella plasticità sinaptica in condizioni non patologiche. Impieghiamo protocolli per coltura microglia dai tessuti neonatali e adulti che mirano a massimizzare il numero di cellule vitali minimizzando variabili confondenti, quali la presenza di altri tipi di cellule del SNC e coltura detriti cellulari. Utilizziamo grandi e facilmente individuabile componenti del sistema nervoso centrale (ad esempio corteccia, segmenti del midollo spinale), che rende l&#39;intero processo fattibile e riproducibile. L&#39;uso di cellula adultas è una valida alternativa all&#39;uso di microglia cerebrale neonatale, come molte patologie indagate interessano principalmente l&#39;postnatale midollo spinale. Questi sistemi di coltura sono anche utile per testare direttamente l&#39;effetto di composti che possono sia inibire o promuovere attivazione della microglia. Dal attivazione della microglia può plasmare gli esiti delle malattie del sistema nervoso centrale adulto, vi è la necessità per i sistemi in vitro in cui microglia neonatale e adulta può essere coltivato e studiato.

<html> Un esempio di cellule microgliali riposo che attivate è mostrato in Figura 1. La microglia sono state visualizzate 24 ore dopo la placcatura (Figura 1a) e presentano ramificate (a riposo) morfologia. Esposizione al reagente adescamento, batteriche lipopolisaccaridi (LPS) provoca cambiamenti nella morfologia della microglia esempio le cellule diventano attivato (Figura 1b). Un esempio di contare le cellule per la placcatura è mostrato...

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

Noi descriviamo i metodi per l&#39;efficiente e rapido isolamento / cultura della microglia praticabile dalla corteccia cerebrale neonatale e midollo spinale adulto. La dissezione e la placcatura di microglia corticale può essere realizzato entro 90 minuti, con la conseguente raccolta microglia in atto ~ 10 giorni dopo la dissezione iniziale.

Coltura Microglia dal Sistema Nervoso Centrale neonatale e adulti

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 Views.  Stony Brook University. Noi descriviamo i metodi per l'efficiente e rapido isolamento / cultura della microglia praticabile dalla corteccia cerebrale neonatale e midollo spinale adulto. La dissezione e la placcatura di microglia corticale può essere realizzato entro 90 minuti, con la conseguente raccolta microglia in atto ~ 10 giorni dopo la dissezione iniziale.

Video: 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.

Isolamento primario Microglia misti da colture cellulari gliali del tessuto neonatale cervello di ratto

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

Ricerca

Immunologia e infezioni

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.

Purificazione e visualizzazione di lipopolisaccaride da batteri Gram-negativi da Hot acquosa-fenolo di estrazione

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).

Isolamento della corticale Microglia con Immunofenotipo conservato e funzionalità dal murini neonati

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.

Sviluppo di un virus System Reporter dell&#39;epatite B per monitorare la fasi iniziali del ciclo di replicazione

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.

Analisi dei Macrofagi Microglia e dei Monociti dal Sistema Nervoso Centrale dalla Citometria del Flusso

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.

Isolando i linfociti del sistema immunitario intestinale piccolo per Mouse

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 dispositivo di posizionamento per il posizionamento dei topi durante la consegna del siRNA intranasale al sistema nervoso centrale

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.

Analisi citometrica del flusso dell'infiltrazione di linfociti nel sistema nervoso centrale durante l'encefalomielite autoimmune sperimentale

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.

Isolamento dei tessuti del sistema nervoso centrale e delle meningi associate per l'analisi a valle delle cellule immunitarie

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.

Protocollo economico ed efficiente per l'isolamento e la coltura di cellule dendritiche derivate dal midollo osseo da topi

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