Questo lavoro è stato supportato dal NIH (R01HL128714 a CF) e (F31DK10236101 a KS) e l&#39;American Heart Association (CF e AG).

Tutti i protocolli di animali e procedure sperimentali sono state approvate dal Comitato Istituzionale Animal Care e Usa (IACUC) dell &#39;Università di Miami Miller School of Medicine. 1. Preparazione <ol><li> mezzi di perfusione epatica di preriscaldamento e fegato digerire media in un bagno d&#39;acqua a 37 ° C. </li><li> Impostare una pompa di perfusione (30 mL / min). precompilare con attenzione il sistema di tubi della pompa con mezzi di perfusione epatica. Evitare eventuali bolle d&#39;aria nel sistema e preparare il microscopio stereotassica. </li><li> Coat piatti di coltura cellulare utilizzando collagene di tipo I secondo il protocollo del produttore. </li></ol> 2. Il recupero del fegato <ol><li> Anestetizzare il mouse donatore utilizzando ossigeno / inalazione isoflurano (isoflurano 4% / 2 L / min di ossigeno). <ol><li> Mantenere anestesia abbassando isoflurano al 2-2,5% o iniettando ketamina (100 mg / kg di peso corporeo per via intraperitoneale) e xilazina (10 mg / kg di peso corporeo intraperitoneally). Continuo anestesia isoflurano è preferito da ketamina abbassa la pressione sanguigna e può causare epatotossicità. </li></ol></li><li> Posizionare il mouse anestetizzato su un tampone assorbente. Fissare le estremità del topo con del nastro adesivo. </li><li> Shave e disinfettare l&#39;addome del mouse ed eseguire una laparotomia ventrale dal pube fino al confine craniale del fegato con le forbici chirurgiche con punte affilate / smussato. Incidere la parete addominale su entrambi i lati, caudale del diaframma. </li><li> Esporre la vena cava inferiore (IVC) spostando attentamente l&#39;intestino al lato destro. sezionare con cura il tessuto grasso intorno alla IVC utilizzando sterili tamponi punta di cotone e pinze punta angolata. </li><li> Incidere il diaframma sovraepatica e aprire la cavità toracica da due incisioni laterali con le forbici chirurgiche sottili con punte taglienti / appuntiti. Legare l&#39;toracica IVC utilizzando seta chirurgica (5/0). </li><li> Cannulate il infrarenale IVC utilizzando un catetere IV schermato, rimuovere con attenzione l&#39;ago,collegare la pompa di perfusione e iniziare perfusione del fegato. Se eseguita correttamente, il fegato dovrebbe iniziare immediatamente a scottare e si gonfiano. </li><li> Transetto della vena porta che consente il drenaggio adeguato dei mezzi di comunicazione di sangue e perfusione con le forbici chirurgiche sottili. </li><li> Profumato fegato con circa 30 ml di fegato mezzi perfusione, e poi passare a fegato digerire i media (30 ml). Spegnere la pompa di perfusione e rimuovere la cannula volta perfusione è completa. </li><li> esporre delicatamente la regione centrale del fegato e individuare il tessuto connettivo che collega i lobi del fegato. Afferra le fibre che collegano centrali e con attenzione sezionare tutti i tessuti che tengono il fegato in atto e rimuovere delicatamente il fegato. </li><li> Trasferire immediatamente il fegato in un tubo conico da 50 ml in polipropilene contenente 15 ml di fegato digerire media. NOTA: Non c&#39;è tempo-linea specifica per questo passo. Dopo perfusione, il fegato rimane in mezzi Digest per il trasporto ad una cappa sterile coltura cellulare. Tutti spassi ubsequent devono essere eseguite in un ambiente sterile. </li></ol> 3. Isolamento e trattamento delle cellule <ol><li> All&#39;interno di una cappa di coltura cellulare, trasferire il fegato da terreni contenenti digestione 50 mL provetta di polipropilene conica in una piastra di coltura tissutale sterili. </li><li> Utilizzando pinze sterili, strappare delicatamente i quattro lobi del fegato. Se eseguita correttamente, medio digestione dovrebbe diventare torbida a causa di isolare epatociti dal fegato. </li><li> Utilizzando una punta 25 mL pipetta sterile, filtrare il mezzo di digestione torbida attraverso un colino 70 micron cella nylon in una nuova provetta conica da 50 ml in polipropilene per rimuovere le particelle di tessuto non digerito e detriti cellulari. </li><li> Ripetere i punti 3.2 e 3.3 con sterile PBS (PBS). Questo aiuta a rimuovere eventuali residui epatociti dal fegato. </li><li> Centrifugare a 50 xg per 2 minuti a 4 ° C. Aspirare il surnatante contenente detriti cellulari in eccesso e delicatamente risospendere le cellule in 25 ml 1x freddoWilliams &#39;Media E. </li><li> Utilizzando un 25 mL punta pipetta sterile, filtrata del risospensione attraverso un nuovo 70 micron colino cella di nylon in un tubo da 50 ml in polipropilene conica per ottenere una sospensione più pulito. </li><li> Ripetere i punti 3.5 e 3.6 per tre lavaggi aggiuntivi utilizzando freddo Williams &#39;Medium E 1x per ottenere una sospensione di cellule più chiara. </li><li> Dopo l&#39;ultimo lavaggio, aspirare il surnatante per rimuovere i detriti delle cellule in eccesso. Risospendere e celle filtranti in 25 ml caldo Williams &#39;Media e 1x che contiene lo scongelamento degli epatociti primari e integratori placcatura attraverso un nuovo 70 micron colino cella di nylon in un tubo da 50 ml in polipropilene conica. </li><li> Contare le celle all&#39;interno della sospensione cellulare 25 mL con un emocitometro. In media si aspettano 15 - 20 milioni di cellule per il fegato adulto. Determinare la vitalità delle cellule utilizzando trypan colorazione blu. </li><li> cellule piastra in 2 ml di Williams &#39;Media e 1x che contiene lo scongelamento degli epatociti primari e supplementi placcatura su un collagene rivestito ce 6-benell cultura del cluster (9 x 10 5 cellule per pozzetto). </li><li> Dopo 4 ore, cambiare media per Williams &#39;Media e 1x che contiene gli integratori di manutenzione epatociti primari. </li><li> Dopo 24 ore, il siero di fame le cellule con medie di Eagle modificato di Dulbecco (DMEM) supporti 1x per 6 ore. </li><li> Trattare cellule con PBS come veicolo, FGF23 (25 ng / mL), LPS (100 ug / mL) o IL-6 (50 ng / ml) in terreno privo di siero e incubare per 24 h. </li></ol> 4. Isolamento di RNA <ol><li> Preparare RNA utilizzando un kit Spin Column commerciale in base alle istruzioni del produttore. </li></ol> 5. Generazione di cDNA da RNA isolato mediante trascrizione inversa <ol><li> Aggiungere 200 ng di campione di RNA isolato dalla provetta. </li><li> Aggiungere 14 ml di-endonucleasi libera H 2 O per PCR tubo dal punto 5.1. </li><li> Aggiungere 4 ml di trascrittasi inversa per provetta PCR dal punto 5.1. </li><li> Risospendere il contenuto con precauzione per fare miscela omogenea; centrifugare delicatamente. </li&gt; <li> Mettere provetta PCR contenente il composto in un termociclatore. Eseguire termociclatore per trascrizione inversa: 1 ciclo di 25 ° C (5 min), 42 ° C (30 min), 85 ° C (5 min) e poi mantenere a 4 ° C. Il prodotto finale sarà il cDNA desiderato. </li></ol> 6. Analisi di celle quantitativa Real-time PCR (qPCR) <ol><li> Preparare la piastra a 96 pozzetti. <ol><li> Mettere tutti i componenti della reazione qPCR su ghiaccio: 1 ml di cDNA, .25 micron di primer in avanti, .25 micron inversione di fondo, 5 ml SYBR Green, 3,5 microlitri PCR acqua di grado per un volume totale di 10 microlitri. Utilizzando SYBR Green, preparare master mix per eseguire tutti i campioni in triplice copia. </li><li> Una volta che il master mix è completo, vortex per rendere omogenea. Aliquota 9 ml di qPCR master mix in ciascun pozzetto di una piastra da 96 WLL. Dopo, aggiungere con cautela 1 ml di cDNA in ciascun pozzetto. Volume di reazione totale sarà di 10 ml. </li><li> Dopo il caricamento è completo, sigillare piastra a 96 pozzetti con otticapellicola adesiva e centrifugare brevemente piastra a 96 pozzetti (50 xg per 1 min). </li></ol></li><li> Campioni eseguito in uno strumento di Real-Time PCR. Mettere sigillato piastra a 96 pozzetti in vero e proprio strumento Time-PCR e campioni Esegui come strumento per le raccomandazioni del produttore. Esempi di cicli standard e veloci sono stati inseriti. <ol><li> Per i parametri di ciclismo normali, utilizzare il seguente: denaturazione iniziale a 94 ° C per 120 s, quindi 40 cicli di 94 ° C per 15 s, 60 ° C per 60 s, e quindi opzionalmente tenere a 4 ° C. </li><li> Per i parametri ciclo rapido: utilizzare il seguente: denaturazione iniziale a 94 ° C per 30 s, quindi 40 cicli di 95 ° C per 5 s, 58 ° C per 15 s, quindi 72 ° C per 10 s. </li></ol></li><li> Fare riferimento al manuale dello strumento per una guida su come analizzare i dati. </li></ol> 7. Analisi dei sopranatanti cellulari di ELISA NOTA: Tutte le operazioni vengono eseguite secondo il protocollo del produttore. </p&gt; <ol><li> Preparazione del campione <ol><li> Dispensare 398 ml di diluente in tubi separati. Pipettare 2 ml del campione surnatante cellulare nella provetta di diluente 398 ml. Ciò fornirà una piega diluizione 200. </li><li> Ripetere il passaggio 7.1.1 per ogni campione da analizzare. </li></ol></li><li> Procedura <ol><li> Pipettare 100 ml di campione diluito 200 volte e uno standard nei pozzetti di una piastra da 96 pozzetti. </li><li> Incubare la piastra a 96 pozzetti a 25 ° C per 45 minuti su un agitatore per micropiastre a 150 rpm. </li><li> Lavare i pozzetti 5 volte con soluzione tampone di lavaggio 1x. Una volta che l&#39;ultimo lavaggio, rimuovere tutta la soluzione di lavaggio residua con carta assorbente. </li><li> Aggiungere 500 ml di reagente HRP-coniugato in tutti i pozzetti. </li><li> Ripetere i punti 7.2.2 - 7.2.3. </li><li> Pipettare 100 ml di tetrametilbenzidina (TMB) dei reagenti in ciascun pozzetto della piastra da 96 pozzetti. </li><li> Mescolare delicatamente piastra a 96 pozzetti a 25 ° C per 20 minuti su una micropiastraagitatore a 150 rpm. Arrestare la reazione aggiungendo direttamente 100 ml di soluzione di stop in ogni pozzetto e mescolare delicatamente. Una volta aggiunto alla soluzione di arresto il colore deve diventare giallo. Questo informa che la soluzione di arresto è stato correttamente miscelata. </li><li> Utilizzo di un lettore di micropiastra, determinare la densità ottica a 450 nm entro i primi 5 minuti. </li></ol></li><li> Calcolo dei risultati <ol><li> Calcolare una tabella di valori di assorbanza media (A 450) per ogni campione e ciascuno standard. </li><li> Montare una curva standard tracciando la media A 450 di ogni standard contro la sua concentrazione standard in ng / mL su un grafico lineare. Consentire l&#39;asse x per rappresentare la concentrazione e l&#39;asse y per rappresentare A 450 valori. </li><li> Utilizzando la media A 450 da ciascun campione diluito, determinare la concentrazione della proteina desiderata in ng / mL dalla curva standard. </li><li> Moltiplicare la concentrazione del campione per la diluizione di fattor. Ciò consentirà per determinare la concentrazione effettiva della proteina desiderata nel campione surnatante cellulare. </li><li> Tracciare la tabella costruita da campioni in un grafico a linee. Se OD 450 valori non rientrano della curva standard, i campioni devono essere diluiti in modo appropriato e ri-testati. </li><li> Normalizzare i risultati a 500.000 cellule. </li></ol></li></ol>

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Isolando epatociti primari da topi è uno strumento veloce, economico e affidabile per studiare le risposte infiammatorie ex vivo. Se eseguita correttamente, i risultati possono essere facilmente generati e riprodotti in modo tempestivo ed economicamente efficiente. I seguenti punti devono essere attentamente valutati per assicurare un isolamento efficace. L&#39;incisione chirurgica e incannulamento della IVC devono essere eseguite in anestesia generale e non dopo l&#39;eutanasia. U...

Malattia renale cronica (CKD) può essere definita come uno stato di infiammazione acuta e cronica 1. Nei pazienti con insufficienza renale cronica, i livelli sierici del fattore di crescita dei fibroblasti ormone phosphaturic 23 (FGF23) progressivo aumento al fine di mantenere l&#39;omeostasi fosfato sierico 2. L&#39;aumento dei livelli sierici di FGF23 sono indipendentemente associati con la morbilità e mortalità cardiovascolare tra i pazienti che iniziano il trattamento di emodialisi 3, 4. Inoltre, diversi studi clinici hanno dimostrato una forte correlazione tra i livelli di FGF23 elevate e livelli sierici di proteina C-reattiva (CRP), interleuchina-6 (IL-6) e Tumor Necrosis Factor α (TNF-alfa) 5, 6. Inoltre, in uno studio sperimentale, abbiamo recentemente dimostrato che FGF23 può indirizzare direttamente epatociti e provocare una risposta infiammatoria aumentando CRP e IL-6 produzione nel fegato 7. Quindi, FGF23 potrebbe agire come un fattore che contribuisce alla circolazione infiammazione sistemica in CKD. Nei primi anni &#39;70, epatociti primari sono stati isolati e studiati per la prima volta 8. Da allora le cellule epatiche in coltura primaria sono stati ampiamente utilizzati per esaminare il trattamento metabolico, la funzione ormonale, il metabolismo dei farmaci e la tossicità, nonché immunità e le risposte infiammatorie 9, 10. Protocolli precedenti hanno descritto principalmente l&#39;isolamento enzimatica di epatociti primari da tessuto epatico umano 11, 12. Mentre un modello eccellente, questo lascia la possibilità di studiare come genetica manipolazione riguarda i meccanismi di segnalazione epatica complessi così come conseguenze funzionali su diversi tipi di stimoli. Nel seguito si descrive l&#39;isolamento di epatociti primari murini. In particolare, l&#39;effetto di diversi mediatori della risposta di fase acuta epatica, come lipopolisaccaride (LPS), IL-6 e FGF23 può essere analizzato in modo facile, veloce e riproducibile 13. Qui, vi presentiamo un protocollo per l&#39;isolamento enzimatica di epatociti di topi adulti, e dimostriamo che induttori stabiliti di infiammazione, come LPS e IL-6, così come mediatori infiammatori innovative come le FGF23, in grado di stimolare direttamente l&#39;espressione e la secrezione di citochine infiammatorie, come la PCR e iL-6 in epatociti coltivati.

<table border="0" cellpadding="0" cellspacing="0" width="924">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">Consumables</td>
			<td style="width:283px;"></td>
			<td style="width:175px;"></td>
			<td style="width:103px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Strainer 70 &#956;m Nylon cell strainer</td>
			<td>Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Insyte Autoguard</td>
			<td>BD</td>
			<td>381412</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 mL Polypropylene Conical Tube</td>
			<td>Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 6-inch Cotton Tipped Applicators&#160;</td>
			<td>Puritan</td>
			<td>806-WC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1cc U-100 Insulin Syringe 28 G 1/2</td>
			<td>Becton Dickinson</td>
			<td>329420</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue Culture Dish 100 x 20 mm Style</td>
			<td>Corning</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 well Cell Culture Cluster</td>
			<td>Costar</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5/0 Black Braided Surgical Silk (100 yards)</td>
			<td>LOOK</td>
			<td>SP115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Minipuls 3 Perfusion Pump</td>
			<td>Gilson</td>
			<td>F155007</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">Surgical Scissers - Sharp/Blunt</td>
			<td>F.S.T.</td>
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			<td height="22" style="height:23px;">Student Tissue Forceps</td>
			<td>F.S.T.</td>
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			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
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			<td height="21" style="height:21px;">Acetic acid solution, 2.0 N</td>
			<td>Sigma</td>
			<td>&#160;&#160;&#160; A8976-100ML</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">Isoflurane, USP 250 mL</td>
			<td>Piramal Healthcare</td>
			<td>&#160;&#160; 66794-013-25</td>
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			<td height="21" style="height:21px;">KetaVed 1000mg/10mL (100mg/mL)</td>
			<td>VEDCO</td>
			<td>&#160;&#160;&#160; 50989-161-06</td>
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			<td height="21" style="height:21px;">Xylazine 100 mg/mL</td>
			<td>AnaSed Injection</td>
			<td>139-236</td>
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			<td height="21" style="height:21px;">Willams&#39; Medium E (1x)</td>
			<td>gibco</td>
			<td>12551-032</td>
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			<td height="21" style="height:21px;">Liver Perfusion Medium (1x)</td>
			<td>gibco</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Liver Digest Medium (1x)</td>
			<td>Life Technologies</td>
			<td>17703034</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Primary Hepatocyte Thawing and Plating Supplements</td>
			<td>Life Technologies</td>
			<td>CM3000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primary Hepatocyte Maintenance Supplements</td>
			<td>Life Technologies</td>
			<td>CM4000</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffer Saline (PBS) pH 7.4</td>
			<td>ThermoFisher scientific</td>
			<td>10010031</td>
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			<td height="21" style="height:21px;">Collagen Type 1</td>
			<td>Corning</td>
			<td>354236</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Trypan Blue Solution</td>
			<td>VWR</td>
			<td>45000-717</td>
			<td></td>
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induction of an inflammatory response in primary hepatocyte cultures from mice

Il fegato svolge un ruolo determinante nella regolazione dell&#39;infiammazione sistemica. Nella malattia renale cronica in particolare, il fegato reagisce in risposta al ambiente uremico, stress ossidativo, endotossiemia e la clearance dell&#39;amlodipina circolante citochine proinfiammatorie producendo un gran numero di proteine ​​della fase acuta. strumenti sperimentali per studiare l&#39;infiammazione e il ruolo di fondo di epatociti sono cruciali per comprendere la regolamentazione e il contributo di citochine epatiche ad una risposta di fase acuta sistemica e uno scenario pro-infiammatoria prolungata, particolarmente in un contesto complicato come la malattia renale cronica. Dal momento che lo studio complessi meccanismi di infiammazione in vivo rimane difficile, e di solito ad alta intensità di risorse richiede l&#39;utilizzo di animali transgenici, epatociti isolati primari forniscono uno strumento robusto per acquisire conoscenze meccanicistici nella risposta della fase acuta epatica. Poiché questa tecnica in vitro offre costi contenuti, alta reproducibilità e la conoscenza tecnica comune, epatociti isolati primari possono anche essere facilmente utilizzato come un approccio di screening. Qui, descriviamo un metodo enzimatico-based per isolare epatociti murini primari, e descriviamo la valutazione di una risposta infiammatoria in queste cellule utilizzando ELISA e quantitativa real-time PCR.

Istologia Immagini luce microscopia rappresentativi di cellule isolate e coltivate primarie sono descritte nella Figura 1A. Immunocytochemical analisi dimostra che epatociti isolati altamente esprimono albumina (rosso) e fattore di crescita dei fibroblasti receptor 4 (FGFR4) (verde). I nuclei sono colorati con 4 &#39;, 6-diamidino-2-phenylindole (DAPI) (blu). (Figura 1B). <p class="jove_step"...

Watch this Scientific Journal Video about Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice at JoVE.com

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

Qui vi mostriamo un approccio enzimatico per isolare epatociti primari da topi adulti, e descriviamo la quantificazione di una risposta infiammatoria con ELISA e PCR in tempo reale.

L&#39;induzione di una risposta infiammatoria in colture primarie epatociti di topi

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

induction-an-inflammatory-response-primary-hepatocyte-cultures-from

Research

JoVE Journal

Immunology and Infection

9.9K Views.  University of Miami Leonard M. Miller School of Medicine.  Qui vi mostriamo un approccio enzimatico per isolare epatociti primari da topi adulti, e descriviamo la quantificazione di una risposta infiammatoria con ELISA e PCR in tempo reale. 

Video: Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

Induzione di ipofisite autoimmune sperimentale nei topi SJL

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

Ricerca

Immunologia e infezioni

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

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

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

Un metodo semplice ed efficace per isolare i macrofagi da Mixed colture primarie di cellule del fegato degli adulti

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)1. The life cycle of HPV is tightly linked to the differentiation of squamous epithelium2. Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production3,4,5. In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras6 and modified by Kopan et al.7, the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies8. Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as well as adenoviruses, parvoviruses, and poxviruses9. Organotypic raft cultures can thus be adapted to examine viral pathogenesis, and are the only means to test novel antiviral agents for those viruses that are not cultivable in permanent cell lines.

An in vitro method to mimic in vivo epithelial differentiation is described. Many viruses target epithelial cells as part of their viral life cycle, and this method provides a means of examining virus:host interactions that more closely resembles that which occurs in vivo. This technique can be used with primary keratinocytes, established cell lines, as well as normal or diseased biopsy tissue.

Generazione di culture Raft organotipiche da cheratinociti umani primari

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

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 ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

Differenziare i ruoli funzionali di espressione genica di cellule immunitarie e non immunitarie in Colite mouse da trapianto di midollo osseo

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

Accelerated diabete di tipo 1 nei topi induzione trasferimento adottivo di cellule CD4 + T Cells diabetogenici

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

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

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

Isolamento e iniezione endovenosa di Murine derivate dal midollo osseo monociti

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

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

Induzione di Murine infiammazione intestinale per trasferimento adottivo di Effector CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Alto</sup&gt; T Cells in topi immunodeficienti

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

Saggi per caratterizzare nuovi endoteliale regolatori coinvolti nella risposta infiammatoria di screening

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

Induzione di paralisi e di lesioni del sistema visivo in topi da specifiche cellule T per neuromielite ottica Autoantigen acquaporina-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

L'induzione di una risposta infiammatoria in colture primarie epatociti di topi

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