Questo lavoro è stato supportato dal NIEHS R01ES014470 (KMZ).

Prima di iniziare questo protocollo, raccogliere topi neonati a giorni postnatali 1-3 (P1-3), in un contenitore sterile nidificato con biancheria gabbia originale per la protezione e calore. E &#39;importante lavorare in modo rapido ed efficiente attraverso questo protocollo per ottimizzare il rendimento della microglia. Si prega di consultare la tabella 1 per la lista completa di reagenti. 1. Preparazione di strumenti, terreni di coltura, e piatti <ol><li> Preparare 10 ml / pup Microglia Complete media (MCM; 1x minimo essenziale del Piano Earle [MEM] supplementato con 1 mM L-glutammina, 1 mM di piruvato di sodio, 0,6% v / v D-(+)-glucosio, 100 pg / ml Penicillina / streptomicina (P / S), 4% v / v siero fetale bovino (FBS), 6% v / v Horse Serum), e aggiungere ad ogni pallone T-75 (1 boccetta / pup); cappuccio e posto orizzontalmente in un incubatore (37 ° C, 5% CO 2) per equilibrare per 1 ora. È essenziale equilibrare questo mezzo beuta (s) prima di iniziare la dissezione. </li><li> Prepare, aliquota, e caldo in 37 ° C bagnomaria MCM per dissezione (6 ml / pup) e in un tubo conico MCM separato per risospensione delle cellule (2 ml / pup). Preparare, aliquote e freddo sul ghiaccio Tritacarne media (MM; HBSS completata con: 100 pg / ml P / S, 0.01 M HEPES, 3 ml / pup), e di dissezione media (DM; MEM supplementato con 100 pg / ml P / S, 2 ml / pup). </li><li> Sterilizzare i seguenti strumenti chirurgici mediante immersione in etanolo al 70% per 10 min: forbice # 1 (1, a decapitare), forcep # 1 (2, per assicurare il naso e rimuovere cortecce), forbice # 2 (1; nonangled, per aprire cuoio capelluto ), forcep # 2 (1; rimuovere cuoio capelluto), forcep # 3 (1; rimuovere cranio), forcep # 4 (2; rimuovere meningi), forbice # 3 (1; tritare tessuto cerebrale). Lasciare asciugare all&#39;aria su un tampone sterile. </li><li> Pulire la postazione di lavoro e la dissezione microscopio orizzontale del flusso d&#39;aria con il 70% di etanolo. </li><li> Dispensare DM in 2 sterili, 100 millimetri piastre di Petri per l&#39;acquisizione di testa (3 ml, 1 piatto / 3 cuccioli) e la dissezione (1 ml;1 piatto / pup; raffreddata in ghiaccio prima di dissezione). Dispensare MM in 1 sterile, 100 millimetri piatto di Petri per tritare il tessuto cerebrale (1 ml, 1 piatto / pup). </li></ol> 2. Cervello Tissue Dissection <ol><li> Prima di eutanasia, igienizzare delicatamente il collo e la testa del cucciolo con il 70% di etanolo utilizzando un tessuto di laboratorio. Con scissor # 1, eutanasia rapidamente pup togliendo la testa, testa di cattura in capsula di Petri precedentemente preparato. Euthanize e completare la dissezione di un cucciolo prima di procedere alla successiva animale. </li><li> Trasferire la testa al piatto dissezione refrigerate; sotto il microscopio di dissezione fissare la testa pizzicando il naso con pinza # 1 (in alternativa, la testa può essere fissata da piercing pinza attraverso la cavità oculare); cuoio capelluto aperto con scissor # 2, creando un Y-cut: iniziare dalla base della testa ed inclinato verso gli occhi. Utilizzare forcep 3 # per rimuovere delicatamente il cuoio capelluto con il peeling laterale, esponendo il cranio. </li><li> Utilizzando forcep # 3, rimuovere delicatamente il tessuto connettivo alla base deltesta. Quando la base del cranio è esposto, accuratamente aperto il cranio infilando un braccio della pinza tra il cervello e il cranio (seguendo la fessura interemisferica); presa; tirare delicatamente il cranio verso l&#39;alto per strappare aperto. </li><li> Disrupt tessuto connettivo sotto il lato ventrale del cervello utilizzando forcep # 1, e successivamente rimuovere il cervello utilizzando lo stesso strumento. Sollevare il cervello su cranio utilizzando una forza verso l&#39;alto sotto la faccia ventrale del cervello. </li><li> Mantenere il cervello nella sua posizione anatomica: lato dorsale verso l&#39;alto per visualizzare le cortecce al microscopio dissezione; fissare il cervello in posizione inserendo punte della pinza nel punto di intersezione del mesencefalo e corteccia; rimuovere completamente meningi corticali con forcep # 4, quindi procedere a rimuovere le meningi ventrali. Una volta che le meningi siano stati completamente asportati, separare le cortecce dal resto del cervello utilizzando pinza # 4. È essenziale rimuovere completamente il tessuto meningeo per massimizzare microglia purezza e yield. </li></ol> 3. Cervello Tissue Mincing <ol><li> Cortecce di trasferimento di preparati in precedenza macinazione piatto, continuare a tritare passo con tecnica sterile in Classe II Sicurezza biologica Gabinetto. </li><li> Tritare il tessuto cerebrale utilizzando forbice # 3; trasferimento di tessuto tritato in una provetta da 15 ml;. Lavare le forbici e tritare piatto con 1 ml di MM ciascuna, raccogliere e dispensare risciacqui nel tubo conico da 15 ml contenente il tessuto tritato Questa fase di risciacquo massimizzerà la resa di microglia garantendo che tutto il tessuto residuo viene trasferito tubo conico. Lascia sospensione tessuto indisturbato per 1-2 minuti, permettendo il tessuto macinata di stabilirsi sul fondo della provetta. Non superare 2 min come MM non contiene nutrienti essenziali. </li><li> Rimuovere con cautela e scartare 2 ml di surnatante con generico 1000 microlitri micropipettatore con un puntale di serie, facendo attenzione a non disturbare il tessuto tritato risolta. </li&gt; <li> Aggiungere 3 ml di MCM al tessuto tritato; triturare il tessuto con forza con un generico 1000 microlitri micropipettatore con un puntale di serie n distinguibile tessuto solido dovrebbe rimanere quando triturazione è completa.. Aggiungere altri 3 ml di MCM con la stessa punta della pipetta, la raccolta del campione triturato residuo dalla punta della pipetta. </li></ol> 4. Crescita delle cellule corticali <ol><li> Centrifugare il tessuto tritato a pellet cellule (215 xg, 5 min; RT, centrifuga clinica con rotore oscillante). Ritorno cellule pellet al gabinetto di sicurezza biologica, rimuovere e scartare il surnatante, risospendere delicatamente il pellet cellulare (2 ml / provetta, MCM), aggiungere la risospensione delle cellule al precedentemente preparato T-75 pallone, tappare il pallone e metterlo in orizzontale in umidificata standard coltura tissutale incubatore (37 ° C, 5% CO 2). Permettono alle cellule di incubare per 24 ore. </li><li> Dopo 24 ore, delicatamente toccare pallone contro il palmo della mano per distruggereframmenti di tessuto. Controllare le cellule prima e dopo aver toccato per garantire la completa rimozione dei detriti. Set pallone verticale; rimuovere ed eliminare media; aggiungere MCM fresco al fondo del pallone (12 ml; 37 ° C); cappuccio e inserire di nuovo nel incubatore, orizzontalmente. </li><li> Controllare le cellule al giorno per la contaminazione e per monitorare la crescita. </li><li> A circa 17-21 giorni in vitro (DIV), le cellule microgliali saranno pronti per il raccolto dalla cultura gliali mista. Per determinare se le cellule sono pronte, esaminare le cellule sotto un microscopio a contrasto di fase invertito. Microglia sono pronti per il raccolto, quando al ~ 40% di confluenza. Essi appaiono come piccoli, luminosi, celle sferiche, crescendo come un monostrato in cima glia altri. </li></ol> 5. Isolamento di Microglia da Mixed Cultura Glial <ol><li> Per isolare microglia, vigorosamente toccare pallone contro il banco di laboratorio. Questa agitazione aiuta microglia, distinta da altre cellule gliali a causa delle proprietà di bassa aderenza della microglia. </li><li&gt; Seal T-75 caps pallone con Parafilm e ruotare le colture gliali miste (200 giri, 37 ° C, 5 ore). utilizzando un nonhumidified agitatore rotante a temperatura controllata Non permettere alle cellule di scuotere per più di 5 ore Effettuare il controllo visivo. che la microglia hanno sollevato dalla superficie pallone dopo 5 ore, utilizzando un microscopio a contrasto di fase invertito. Microglia sono luminose, sferico, free-floating cellule. Ci sarà uno strato residuo di astrociti e oligodendrociti aderenti al fondo del pallone. </li><li> Raccogliere i media surnatante contenente microglia in una provetta da centrifuga conica sterile; microglia a pellet (215 xg, 5 min, oscillanti rotore secchio). </li><li> Risospendere il pellet microglia nel warm microglia crescita medio (MGM; MEM supplementato con: 1 piruvato di sodio mM, 0,6% v / vglucose, 1 mM L-glutammina, 100 pg / ml P / S, 5% v / v di FBS; ~ 2 ml / beuta), determinare la concentrazione cellulare mediante un emocitometro e piastra 1 x 10 5 cellule / pozzetto in un formato di piastra da 24 pozzetti sullaplastica o sterili scivola copertura in vetro (500 microlitri / e; MGM). Utilizzando questa procedura il rendimento medio microglia è di 7,5 x 10 5 cellule / pup corteccia. </li><li> 24 ore di post-placcatura, rimuovere tutti i mezzi di comunicazione e delle cellule refeed con MGM fresco (37 ° C) per rimuovere le cellule galleggianti e detriti. Questo passaggio è fondamentale alla sopravvivenza delle cellule e mantenere microglia quiescenti. Le cellule possono essere utilizzati per la sperimentazione subito dopo rialimentazione. </li></ol> 6. Trattamenti Esempio <ol><li> Esporre le cellule a 10 ng / ml di Pam 3 CSK 4 (Pam), 10 ng / ml di lipopolisaccaride (LPS) o di controllo del veicolo; incubare per 2 o 24 ore in incubatore umidificato (37 ° C, 5% CO 2). </li></ol> 7. Analizzando Microglia per la funzionalità ex vivo tramite immunofluorescenza Imaging e Immunocytochemistry <ol><li> Dopo il trattamento, mezzi di coltura cellulare raccolto in provette per microcentrifuga (1,5 ml) e centrifuge utilizzando una microcentrifuga da tavolo (2 min; 900 xg) per cancellare i media. Trasferire il supernatante in una provetta per microcentrifuga pulita; dosaggio immediatamente, o conservare a -20 ° C fino al momento dell&#39;uso. Analizzare concentrazione di TNF-α in coltura cellulare surnatante tramite disponibile in commercio del mouse-TNF α kit ELISA. </li><li> Lavare le cellule con tampone fosfato (PBS), e fissare le cellule con il 4% (w / v) di paraformaldeide / 4% (w / v) di saccarosio / PBS pH 7,4 (4% PFA, 15 min; RT). Dopo la fissazione, lavare le cellule con PBS per 5 minuti agitando delicatamente su un agitatore orbitale. <ol><li> Immunofluorescenza / Immunocytochemistry. Dopo la fissazione e PBS lavare, processo microglia per immunocitochimica. Tutte le fasi effettuate con un leggero scuotimento. Permeabilize cellule in PBS/0.1% triton X-100; cellule blocchi in siero normale di capra PBS/10% per un&#39;ora. Incubare le cellule O / N a 4 ° C con coniglio anti-NF-kB anticorpo (p65 subunità, 1/1, 250) o coniglio anti-Iba-1 anticorpo (1/750) nel tampone di bloccaggio. Visivoize antigene: anticorpo seguente incubazione con fluorescenza di capra coniugato anti-IgG di coniglio anticorpo secondario (1 ora, 1/1, 000). Rimuovere l&#39;anticorpo secondario non legato mediante lavaggio con PBS/0.1% triton X-100 (3x 5 min). </li><li> Cellule macchia contatore con 4 &#39;,6-Diamidino-2-phenylindole / PBS (DAPI; 0,1 mg / ml). Per visualizzare nucleo della cellula Tutti microglia derivati ​​da CX3CR1-GFP + / - topi esprimono GFP. Utilizzare un filtro di lunghezza d&#39;onda di 350 nm per visualizzare DAPI, un filtro di lunghezza d&#39;onda di 488 nm per visualizzare GFP, ed un filtro di lunghezza d&#39;onda 594 nm di visualizzare p65 e Iba-1 immunocomplessi. </li></ol></li><li> Montare il coperchio scivola usando la soluzione di montaggio acquosa e visualizzare microglia usando la microscopia a fluorescenza. </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>

Gli autori dichiarano che la ricerca è stata condotta in assenza di rapporti commerciali o finanziari che potrebbero essere interpretate come un potenziale conflitto di interessi.

Il presente procedimento offre un metodo efficace per l&#39;isolamento di microglia corticali di topi neonati. Questa procedura ha un duplice vantaggio di 1) conservare immunofenotipo microglia e funzionalità, come determinato mediante imaging fluorescente, immunocitochimica e ELISA, e, 2) permettendo microglia di maturare in presenza di altre cellule gliali (astrociti e oligodentrocytes) prima isolamento, che può facilitare importanti interazioni cellula-cellula durante il periodo di coltura maturazione gliale. È im...

Cellule microgliali, i macrofagi sorveglianza del parenchima SNC, comprendono circa il 12% della popolazione totale di cellule del cervello dei mammiferi adulti. Microglia sono derivati ​​dal sacco vitellino mieloidi precursori delle cellule e variano in densità cellulare e morfologia in diverse regioni cytoarchitectural all&#39;interno del SNC adulto 1-5. In un cervello adulto sano, microglia sono piccoli, ramificati o polari cellule con raffinati processi dinamici. In contrasto con macrofagi morfologia periferico, microglia dimostrano una quiescente, basso profilo fenotipo in cervelli sani che possono apparire come l&#39;inattività cellulare, tuttavia, vivo studi di imaging in dimostrano che i processi microgliali si estendono in modo dinamico e ritraggono per monitorare il loro microambiente in un modo che ricorda &quot; campionamento e rilevamento &quot;6,7. Microglia sono altamente e differenzialmente sensibili alle alterazioni ambientali e patofisiologici nel cervello, commutazioneda una surveillant a un effettrici stati, rispettivamente statali comunemente considerato come il loro riposo e attivato. Questo interruttore di attivazione può essere mediata da un blocco del recettori di membrana pattern recognition (PRR), come i recettori toll-like (TLR), che rispondono a modelli molecolari associati ai patogeni (PAMP), lipoproteine ​​vale a dire-batteriche e virali derivati , acidi nucleici, carboidrati e 8-11. Oltre a PAMPs, PRRs hanno dimostrato di indurre l&#39;attivazione della microglia contro sterili, molecole patogene conosciute come modelli molecolari pericolo / danno associato (smorza), che rappresentano una perturbazione nel sistema nervoso centrale dell&#39;omeostasi, come danno cellulare 12-16. Una volta inserita, PRRs avviare una cascata di segnalazione intracellulare che si traduce in cambiamenti nella morfologia cellulare e l&#39;espressione genica microglia, in particolare, microglia attivata adattare un fenotipo ameboide-like, traslocare la p65 NF-kB subunità (p65) al nucleo della cellula, e il potenziamento della produttoriction e la secrezione di citochine proinfiammatorie, come fattore di necrosi tumorale-α (TNF-α), interleuchina-1 β (IL-1β), insieme con le specie reattive dell&#39;ossigeno (ROS) 16-24. Sebbene integrale nella risposta immunitaria innata del sistema nervoso centrale, queste molecole secrete sono stati trovati anche ad aumentare lo stress ossidativo neuronale, inducendo in tal modo aggravando e neurodegenerazione in stati malati come il Parkinson e il morbo di Alzheimer 25-29. Tuttavia, i meccanismi di attivazione della microglia in stati patologici non sono del tutto chiare. Pertanto, l&#39;isolamento di microglia è un potente strumento di indagine in tali processi biologici, come molti nelle caratteristiche vivo di attivazione della microglia può essere ricapitolato in cultura. Sono disponibili diversi metodi per isolare microglia, compreso l&#39;isolamento con gradiente Percoll seguente digestione enzimatica del tessuto CNS 30,31. Tuttavia, la digestione enzimatica può alterarel&#39;immunofenotipo delle cellule riducendo superficie cellulare espressione dell&#39;antigene 32, e si traduce in quantità di cellule per animale inferiore rispetto al metodo qui descritto. In particolare, si segnala un rendimento medio microglia per pup corteccia di 7,5 x 10 5 cellule, mentre precedentemente riportato metodi di isolamento da tutto CNS con gradiente resa Percoll 3-5 x 10 5 cellule 30,33,34. Il presente procedimento elude l&#39;uso di enzimi digestivi isolando microglia in base alle loro proprietà a bassa aderenza, preservando così l&#39;immunofenotipo microglia e funzionalità. Nel presente studio, si descrive l&#39;isolamento di cellule microgliali da colture gliali miste derivate da neonati eterozigoti CX3CR1-GFP (CX3CR1-GFP + / -), e C57BL / 6 cortecce murini tramite agitazione meccanica su un agitatore rotante, una proroga di precedenza metodo pubblicato 24,35. Utilizziamo il primo ceppo di topi per una facile visualizzazione di microglia, comequesti topi esprimono GFP sotto il controllo del locus endogeno CX3CR1 - un promotore specifico monociti 36-38. Questo metodo produce colture microgliali altamente puri con una preservata immunofenotipo ex vivo, come dimostrato dai cambiamenti morfologici, traslocazione nucleare di p65, e la secrezione di TNF-α quando provocati con lipopolisaccaride batterico (LPS) o Pam 3 CSK 4, TLR4 e TLR1 / 2 agonisti rispettivamente.

<table border="0" cellpadding="0" cellspacing="0" width="680">
	<colgroup>
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	</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>
</table>

isolation of cortical microglia with preserved immunophenotype and functionality from murine neonates

Isolamento di microglia dal tessuto del sistema nervoso centrale è un potente strumento di indagine utilizzato per studiare biologia microglia ex vivo. Il presente metodo dettaglia un procedimento per l&#39;isolamento di microglia da cortecce neonatali murini mediante agitazione meccanica con un agitatore rotante. Questo isolamento microglia metodo rendimenti microglia corticali purissime che presentano caratteristiche morfologiche e funzionali indicative di microglia quiescenti in condizioni non patologiche normali in vivo. Questa procedura preserva anche la immunofenotipo microglia e funzionalità biochimica come dimostra l&#39;induzione di modificazioni morfologiche, traslocazione nucleare della subunità p65 di NF-kB (p65), e la secrezione della citochina proinfiammatoria segno distintivo, fattore di necrosi tumorale-α (TNF-α ), su lipopolisaccaride (LPS) e Pam 3 CSK 4 (Pam) sfide. Pertanto, l&#39;attuale procedura di isolamento conserva l&#39;immunofenotipo sia quiescente e attivamicroglia vato, fornendo un metodo sperimentale di investigare microglia biologia ex vivo condizioni.

Conservando la immunofenotipo e la funzionalità della microglia ex vivo durante l&#39;isolamento è fondamentale per essere in grado di utilizzare queste cellule come un modello di indagine per la biologia della microglia. Al fine di dimostrare la conservazione di successo della microglia immunofunctionality con il metodo attuale, abbiamo isolato microglia corticali da neonati P3 (CX3CR1-GFP + / - e C57BL / 6) e le colture trattate sia con LPS o Pam. Come illustrato nell...

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

Una chiave per il successo indagini di biologia microglia è la conservazione dei immunofunction microglia ex vivo durante l&#39;isolamento dal tessuto del sistema nervoso centrale. Isolare microglia via rotanti risultati si stringono in colture cellulari altamente puri e immunofunctional come valutato da immagini fluorescenti, immunocitochimica, ed ELISA dopo l&#39;attivazione della microglia con il lipopolisaccaride stimoli pro-infiammatori (LPS) e Pam 3 CSK 4 (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 ...

isolation-cortical-microglia-with-preserved-immunophenotype

Research

JoVE Journal

Immunology and Infection

15.6K Views.  Georgetown University Medical Center.  Una chiave per il successo indagini di biologia microglia è la conservazione dei immunofunction microglia ex vivo durante l'isolamento dal tessuto del sistema nervoso centrale. Isolare microglia via rotanti risultati si stringono in colture cellulari altamente puri e immunofunctional come valutato da immagini fluorescenti, immunocitochimica, ed ELISA dopo l'attivazione della microglia con il lipopolisaccaride stimoli pro-infiammatori (LPS) e Pam 3

Video: 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.

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

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.

Isolamento, elaborazione ed analisi dei murini gengivali

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.

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 ...

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.

Isolamento di cellule murine Linfonodo stromali

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.

L&#39;isolamento dei leucociti di tessuti murini all&#39;interfaccia materno-fetale

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.

L&#39;isolamento e l&#39;attivazione di linfociti murini

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.

Isolamento delle vescicole extracellulari dal fluido di lavaggio broncoalveolare murino usando una tecnica di centrifugazione di ultrafiltrazione

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.

Applicazione di perturbazioni costanti simili a massaggi sui vitelli per topi e monitoraggio dei conseguenti cambiamenti di pressione intramuscolare

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.

Isolamento delle cellule mononucleari Di Lamina propria dal colon Murine utilizzando 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 ...

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.

Isolamento concomitante di astrociti primari e microglia per l'infezione da parassiti di Protozoi

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