OCS è supportato da un NIH Ruth L. Kirschstein NRSA (F32 DK091040), AK è sostenuto da una borsa di studio post-dottorato dalla American Diabetes Association (7-10-MI-05), e AHH è supportato da un American Heart Association Investigator Award Fondata (12EIA8270000). Esperimenti di citometria a flusso sono stati eseguiti nel VMC Citometria a Flusso risorsa condivisa. Il VMC Citometria a Flusso risorsa condivisa è supportata dalla Vanderbilt Digestive Disease Research Center (DK058404).

1. Reagenti e accessori per la Prima di iniziare questo protocollo sperimentale, preparare i seguenti reagenti: <ol><li> 70% di etanolo </li><li> PBS 1X </li><li> 1X DPBS (senza Ca e Mg) integrati con 0,5% BSA </li><li> FACS buffer: 1X DPBS (senza Ca e Mg), 2 mM EDTA, e l&#39;1% FCS </li><li> Tampone ACK: 150 mM NH 4 Cl, 10 mM KHCO 3, e 0,1 mM Na 2 EDTA in acqua </li></ol> 2. Raccolta e Preparazione del tessuto adiposo <ol><li> Euthanize topi secondo procedure IACUC approvati specifici per ogni istituzione. </li><li> Bagnare completamente il pelo con il 70% di etanolo. </li><li> Fare un&#39;incisione a livello del processo xifoideo (parte inferiore dello sterno) e aprire la cavità toracica per esporre il cuore, facendo attenzione a non recidere le principali vasi sanguigni. </li></ol> Nota: A questo punto, è anche utile per lasciare il diaframma intatto il piùpossibile e tagliare una tacca dal lato destro della gabbia toracica per permettere al sangue di fluire e perfusato dalla cavità toracica. <ol start="4"><li> Agganciare l&#39;atrio destro per permettere al sangue e perfusato per sfuggire al sistema circolatorio. </li></ol> Nota: Quando si tratta di topi obesi, eccesso pericardico AT può avere bisogno di essere rimosso per consentire l&#39;accesso al cuore. <ol start="5"><li> Afferrare il cuore con una pinza e inserire delicatamente un ago nel ventricolo sinistro attraverso l&#39;apice. Perfusione lentamente il mouse con 15 ml di PBS sterile. </li></ol> Nota: ridurre il tasso di perfusione se i polmoni iniziano a riempirsi ed espandersi. <ol start="6"><li> Aprire la cavità peritoneale e rimuovere i cuscinetti di grasso perigonadal con attenzione per evitare eventuali tessuti gonadici. </li><li> Mettere cuscinetti di grasso in una pesata barca sul ghiaccio contenente 2 ml di 1X DPBS (senza Mg o Ca) integrata con 0,5% BSA, e tritare la AT in pezzi pregiati. </li></ol>Nota: Limitare la quantità di AT per pesare barca a 1,2 g. Se la quantità di AT supera 1,2 g, dividerlo equamente tra due barche a pesare. <ol start="8"><li> Conservare i campioni in ghiaccio e preparare 3 ml di soluzione di collagenasi digest per AT campione costituito da 1X DPBS integrato con 0,5% BSA, 10 mM CaCl 2, e 4 mg / ml di collagenasi di tipo II. </li></ol> 3. Collagenasi Digestione <ol><li> Transfer per provette coniche da 50 ml versando l&#39;omogeneizzato e risciacquo la barca pesare con 1 ml DPBS (0,5% BSA) e 3 ml di soluzione di collagenasi II digest. </li><li> Incubare a omogeneizzato in uno shaker di rotazione (200 rpm) a 37 ° C per 20 min. </li><li> Aggiungere 10 ml DPBS (0,5% BSA) per tubi conici e posto sul ghiaccio. </li><li> Triturare omogeneizzato numerose volte utilizzando una pipetta sierologica 10 ml, e passare sospensioni cellulari attraverso il filtro 100 micron in un nuovo tubo da 50 ml. </li><li> Centrifugare la sospensione cellulare a 500 xg per 10 minuti a 4 &amp; dad esempio, C. </li><li> Decantare il surnatante e risospendere SVF pellet cellulare in 3 ml di tampone ACK per la lisi degli eritrociti contaminanti. </li><li> Aggiungere 12 ml di tampone FACS sospensione cellulare e centrifugare a 500 xg per 10 min a 4 ° C. </li><li> Decantare il surnatante e risospendere SVF pellet di cellule in tampone FACS. </li></ol> Nota: utilizzare la dimensione del pellet cellulare come guida per quanto tampone FACS per risospendere in Se il pellet cellulare copre il fondo della provetta 50 ml conica, utilizzare 0,5-1 ml tampone FACS, altrimenti, risospendere in 0.25-0.5 ml. <ol start="9"><li> I campioni su ghiaccio e preparare 1:10 aliquote di diluizione di ogni campione per il conteggio delle cellule di miscelazione 40 microlitri di buffer FACS, 50 soluzione blu tripano microlitri (0,2%), e 10 microlitri di sospensione cellulare. </li><li> Contare le cellule vitali sulla base di blu tripano esclusione e diluire sospensioni di cellule ad una concentrazione finale di 5-10 x 10 6 cellule / ml. </li></ol> 4. La colorazione degli antigeni di superficie <ol&gt; <li> Aggiungere antitopo CD16/CD32 anticorpo (Fc block) ad una concentrazione finale di 0,5-1 μg/10 6 cellule, e incubare in ghiaccio per 10 min. </li><li> Campioni di trasferimento (≥ 10 6 cellule) a 12 x 75 mm provette a fondo tondo di polistirolo. Preparare tubi separati se analizzando bancomat e le cellule T, e unire le celle in più per preparare un adeguato numero di tubi per accogliere il risarcimento richiesto e uno (FMO) controlli fluorescenza modificati meno. </li></ol> Nota: Per fare un esempio, nel quantificare la percentuale di sportelli automatici in base a F4/80 e CD11b, i seguenti compensi e controlli FMO dovrà essere preparato: - Avviare l&#39;elaborazione (cellule) - DAPI o ioduro di propidio (PI) macchia singole (cellule, non aggiungono colorante vitalità fino al punto 5.1) - F4/80 APC (cellule o sfere di compensazione) singoli macchia - CD11b FITC cellule o singole macchia (sfere di compensazione) - FMO 1 (celle): Rat IgG2a κ isotipo di controllo APC + CD11b FITC + colorante vitalità (aggiunto a passo 5.1) - FMO 2 (celle): F4/80 APC + Rat IgG2b κ isotipo di controllo FITC + colorante vitalità (aggiunto a passo 5.1) <ol start="3"><li> Aggiungi anticorpi primari fluoroforo-coniugato e / o controlli isotipo alla concentrazione adeguata (vedi Tabella di reagenti e materiali). </li><li> Proteggere i campioni dalla luce e incubare a 4 ° C per 30 min. </li><li> Aggiungere 2 ml di tampone FACS sospensione cellulare e centrifugare a 500 xg per 5 minuti a 4 ° C. </li><li> Decantare il surnatante e risospendere SVF pellet di cellule in 2 ml di tampone FACS. </li><li> Centrifugare la sospensione cellulare a 500 xg per 5 minuti a 4 ° C e risospendere SVF pellet cellulare in ≥ 400 microlitri di buffer FACS. </li><li> Trasferire campioni da 12 x 75 mm in polistirolo tubi fondo sferico dotato di un 35 top tube colino cellula micron.</li><li> Proteggere dalla luce e conservare i campioni a 4 ° C fino al momento dell&#39;analisi FACS. </li></ol> Nota: Per risultati ottimali cellule devono essere analizzati immeadiately, tuttavia, l&#39;analisi FACS con questo protocollo è stato condotto con successo su cellule marcate memorizzati nel tampone FACS per 1-2 ore a 4 ° C. Se le cellule devono essere fissati per aumentare il tempo di conservazione, cellule marcate possono essere fissate con paraformaldeide al 2% a 4 ° C per 24 ore prima dell&#39;analisi FACS. Aziende anticorpi suggeriscono che le cellule marcate possono essere conservati fino a una settimana, ma questo non è stato testato nel contesto di questa procedura. 5. FACS analisi <ol><li> Prima di analisi FACS, aggiungere colorante vitalità di campioni e controlli adeguati per consentire la discriminazione delle cellule vive / morte. </li></ol> Nota: Numerose coloranti vitalità sono commercialmente disponibili, ma DAPI e PI sono raccomandati seconda della eccitazione / emissione profiles degli anticorpi fluoroforo-coniugati utilizzati. DAPI e propidio ioduro vengono aggiunti a ciascun campione ad una concentrazione finale di 0,2 mg / ml. <ol start="2"><li> Utilizzare un campione di controllo negativo senza macchia, preferibilmente cellule isolate dal tessuto stesso, come i campioni sperimentali, per regolare side scatter (SSC) e forward scatter (FSC) in modo che la popolazione cellulare (s) di interesse sono in scala. </li></ol> Nota: Per l&#39;analisi delle cellule AT SVF, si consiglia di SSC viene visualizzato in una scala logaritmica contro FSC in una scala lineare. L&#39;uso di una scala logaritmica per SSC è particolarmente importante quando si analizzano ATM, che sono spesso molto grandi e granulare. <ol start="3"><li> Disegnare un cancello diffusione luminosa iniziale in base al tipo di cellula (s) da analizzare, e regolare il tubo fotomoltiplicatore (PMT) guadagno in modo che le cellule non colorate sono all&#39;estrema sinistra di un istogramma solo parametro (approssimativamente centrato su 10 2) per i canali appropriati. &lt;/ Li&gt; </ol> Nota: Si raccomanda di linfociti e macrofagi (o altre cellule mieloidi) essere analizzati separatamente a causa di differenze di autofluorescenza. <ol start="4"><li> Utilizzare singoli controlli macchiati o anticorpo di cattura sfere di compensazione per eseguire la compensazione multi-colore. </li></ol> Nota: Si consiglia l&#39;uso di sfere di compensazione, tuttavia, la compatibilità di ogni anticorpo deve essere garantita. Ad esempio, sfere di compensazione potrebbero non cross-reagiscono con anticorpi di coniglio, nel qual caso le cellule isolate sono tenuti ad ottenere un adeguato controllo singolo macchia. <ol start="5"><li> Impostare cancelli sperimentali basati sui controlli modificati FMO. </li><li> Impostare il citometro di flusso per raccogliere il numero appropriato di eventi basati sulla prevalenza della popolazione di interesse e registrare i dati sperimentali. </li><li> Esportare i file FCS di dati per l&#39;analisi offline. Ci sono numerosi programmi disponibili per il flusso cytometrl&#39;analisi dei dati y. Si consiglia Cytobank, una piattaforma web-based che permette investigatores per memorizzare e analizzare i dati e generare dati da qualsiasi computer con accesso a internet 18. Inoltre, Cytobank offre la possibilità di rendere pubblici i dati accessibili o limitare l&#39;accesso a collaboratori. </li></ol>

<ol>
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P., 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=Obesity+is+associated+with+macrophage+accumulation+in+adipose+tissue.">Obesity is associated with macrophage accumulation in adipose tissue.</a> The Journal of Clinical Investigation. 112, 1796-1808 (2003).</li><li>Xu, H., 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=Chronic+inflammation+in+fat+plays+a+crucial+role+in+the+development+of+obesity-related+insulin+resistance.">Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance.</a> The Journal of Clinical Investigation. 112, 1821-1830 (2003).</li><li>Mathis, D., Shoelson, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunometabolism:+an+emerging+frontier.">Immunometabolism: an emerging frontier.</a> Nature Reviews. 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Non abbiamo nulla da rivelare.

Crescente interesse per il ruolo del sistema immunitario nelle conseguenze metaboliche dell&#39;obesità ha portato all&#39;uso diffuso di citometria a flusso per caratterizzare cellule immunitarie del AT. Anche se il protocollo esatto varierà tra laboratori sulla base della loro esperienza e le attrezzature disponibili, i passaggi critici includono collagenasi digestione, centrifugazione differenziale, e la cellula antigene di superficie di etichettatura. Lo scopo del presente articolo è di fornire un protocollo dett...

Storicamente, il tessuto adiposo (AT) è stato visto come un organo inerte di stoccaggio dei lipidi, che si espande e si contrae in risposta al bilancio energetico. Ora capiamo che AT rappresenta un organo endocrino dinamica che secerne attivamente una serie di ormoni che influenzano direttamente il comportamento alimentare e l&#39;omeostasi del glucosio sistemico. Inoltre, negli ultimi dieci anni c&#39;è stato un crescente apprezzamento per le numerose popolazioni di cellule immunitarie che risiedono nella AT frazione stromale vascolare (SVF), così come il loro contributo alla AT omeostasi. La possibilità di separare la AT adipociti e SVF utilizzando un collagenasi digest seguita da centrifugazione differenziale è stato descritto da Rodbell nel 1964 1. Collagenasi II è più spesso utilizzato per la separazione degli adipociti e SVF a causa di manutenzione di adipociti recettori dell&#39;insulina 1. All&#39;inizio, frazionamento enzimatica di AT è stato principalmente impiegato per studiare adipocyte metabolismo e per isolare preadipocytes. Più recentemente, questa tecnica, combinata con l&#39;ampia disponibilità di citofluorimetri e il numero sempre crescente di anticorpi coniugati fluoroforo disponibili in commercio, ha facilitato la caratterizzazione di AT cellule immunitarie. Sebbene la presenza di cellule immunitarie nella infiammato AT era stato descritto in precedenza 2, gli articoli fondamentali per Weisberg et al. e Xu et al. pubblicato nel 2003, furono i primi a documentare l&#39;accumulo di AT macrofagi (ATM) in obesità, che secernono citochine infiammatorie e correlare con AT-specifica e sistemica insulino-resistenza 3,4. Queste osservazioni hanno fornito la base di un nuovo campo di indagine recentemente coniato, &quot;immunometabolism,&quot; 5 e sono state seguite da studi che implicano varie popolazioni di cellule immunitarie, comprese le cellule dendritiche 6, mastociti, cellule T 7 8 -10, cellule B, cellule NKT 11 12 13, eosinofili e neutrofili 14,15 nello sviluppo dell&#39;obesità associata resistenza all&#39;insulina. L&#39;obiettivo di questo articolo è di fornire una descrizione dettagliata della tecnica digest collagenasi utilizzata per isolare le cellule del AT SVF e caratterizzare bancomat e AT cellule T mediante citometria a flusso. Questo protocollo è stato ottimizzato per mouse, tuttavia, gli spettatori possono trarre beneficio dalla lettura di un ottimo articolo che fornisce ampi dettagli sulla ottimizzazione di questa tecnica per l&#39;uomo a 16 anni. I destinatari di questo articolo include investigatori con limitata esperienza di lavoro con il mouse e l&#39;esecuzione di citometria a flusso. Diverse considerazioni pratiche per il bilanciamento resa cellulare e la vitalità con il tempo e le risorse sono presentate così come controlli di citometria a flusso ottimali per la caratterizzazione di AT popolazioni di cellule immunitarie. Oltre al nostro protocollo, i lettori sono referred un recente articolo JoVE da Basu et al. per una eccellente discussione di alcuni aspetti tecnici della citometria a flusso per comprendere adeguati controlli e compensazioni 17.

<table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalog #</td>
 <td></td>
 </tr>
 <tr>
 <td>DPBS (no Ca or Mg) 10 x 500 ml</td>
 <td>Life Technologies</td>
 <td>14190-250</td>
 <td></td>
 </tr>
 <tr>
 <td>DAPI</td>
 <td>Life Technologies</td>
 <td>D3571</td>
 <td></td>
 </tr>
 <tr>
 <td>BSA</td>
 <td>Sigma</td>
 <td>A2153-100G</td>
 <td></td>
 </tr>
 <tr>
 <td>collagenase</td>
 <td>Sigma</td>
 <td>C6885-5G</td>
 <td></td>
 </tr>
 <tr>
 <td>propidium iodide solution</td>
 <td>Sigma</td>
 <td>P4864-10 ml</td>
 <td></td>
 </tr>
 <tr>
 <td>stable stack 20 microliter</td>
 <td>Rainin</td>
 <td>SS-L10</td>
 <td></td>
 </tr>
 <tr>
 <td>20 microliter filter tips</td>
 <td>Rainin</td>
 <td>SRL10F</td>
 <td></td>
 </tr>
 <tr>
 <td>stable stack 250 microliter tips</td>
 <td>Rainin</td>
 <td>SS-L250</td>
 <td></td>
 </tr>
 <tr>
 <td>1000 microliter tips</td>
 <td>Rainin</td>
 <td>GPS-L1000</td>
 <td></td>
 </tr>
 <tr>
 <td>1000 microliter filter tips</td>
 <td>Rainin</td>
 <td>GP-L1000F</td>
 <td></td>
 </tr>
 <tr>
 <td>250 microliter Filter Tips</td>
 <td>Rainin</td>
 <td>SR-L200F</td>
 <td></td>
 </tr>
 <tr>
 <td>FC Block</td>
 <td>BD Biosciences</td>
 <td>553142</td>
 <td></td>
 </tr>
 <tr>
 <td>fisher 100mn strainers</td>
 <td>Fisherbrand</td>
 <td>22-363-549</td>
 <td></td>
 </tr>
 <tr>
 <td>medium weigh dish</td>
 <td>Fisherbrand</td>
 <td>02-202B</td>
 <td></td>
 </tr>
 <tr>
 <td>aluminum foil</td>
 <td>Fisherbrand</td>
 <td>1213100</td>
 <td></td>
 </tr>
 <tr>
 <td>mincing scissors</td>
 <td>Fisherbrand</td>
 <td>089531B</td>
 <td></td>
 </tr>
 <tr>
 <td>Vortex</td>
 <td>Fisherbrand</td>
 <td>2215365</td>
 <td></td>
 </tr>
 <tr>
 <td>50 ml conical tubes</td>
 <td>BD Falcon</td>
 <td>14-959-49A</td>
 <td></td>
 </tr>
 <tr>
 <td>filter top FACS tubes</td>
 <td>BD Falcon</td>
 <td>352235</td>
 <td></td>
 </tr>
 <tr>
 <td>10 ml pipette case 200</td>
 <td>BD Falcon</td>
 <td>1367520</td>
 <td></td>
 </tr>
 <tr>
 <td>round bottom tubes</td>
 <td>BD Falcon</td>
 <td>352058</td>
 <td></td>
 </tr>
 <tr>
 <td>5 ml syringe</td>
 <td>BD Falcon</td>
 <td>309646</td>
 <td></td>
 </tr>
 <tr>
 <td>V Bottom Plates</td>
 <td>Costar</td>
 <td>07-200-107</td>
 <td></td>
 </tr>
 <tr>
 <td>transfer bulb pipette</td>
 <td>Thermo Scientific</td>
 <td>13-711-22</td>
 <td></td>
 </tr>
 <tr>
 <td>Shaker</td>
 <td>Thermo Scientific</td>
 <td>11 676 071</td>
 <td></td>
 </tr>
 <tr>
 <td>Adhesive mat</td>
 <td>Thermo Scientific</td>
 <td>1368750</td>
 <td></td>
 </tr>
 <tr>
 <td>Cell Culture Centrifuge</td>
 <td>Sorvall</td>
 <td>75253839</td>
 <td></td>
 </tr>
 <tr>
 <td>Adapters</td>
 <td>Sorvall</td>
 <td>75003723</td>
 <td></td>
 </tr>
 <tr>
 <td>Rat anti-mouse CD16/CD32</td>
 <td>BD Biosciences</td>
 <td>553142</td>
 <td>Concentration: 0.5 - 1 &mu;g/ 106 cells</td>
 </tr>
 <tr>
 <td>Rat anti-mouse F4/80</td>
 <td>eBioscience</td>
 <td>17-4801</td>
 <td>Fluorophore conjugate: APC 
 Concentration: 0.2 &mu;g/ 106 cells 
 Isotype control catalog number: 17-4321</td>
 </tr>
 <tr>
 <td>Rat anti-mouse CD11b</td>
 <td>eBioscience</td>
 <td>11-0112</td>
 <td>Fluorophore conjugate: FITC 
 Concentration: 0.5 &mu;g/ 106 cells 
 Isotype control catalog number: 11/1/4031</td>
 </tr>
 <tr>
 <td>Armenian Hamster anti-mouse CD11c</td>
 <td>eBioscience</td>
 <td>12-0114</td>
 <td>Fluorophore conjugate: PE 
 Concentration: 0.8 &mu;g/ 106 cells 
 Isotype control catalog number: Dec-88</td>
 </tr>
 <tr>
 <td>Goat anti-mouse MGL1/2</td>
 <td>R&amp;D Systems</td>
 <td>FAB4297P</td>
 <td>Fluorophore conjugate: PE 
 Concentration: 0.1 &mu;g/ 106 cells 
 Isotype control catalog number: IC108P</td>
 </tr>
 <tr>
 <td>Goat anti-mouse CD206</td>
 <td>R&amp;D Systems</td>
 <td>FAB2535P</td>
 <td>Fluorophore conjugate: PE 
 Concentration: 0.1 &mu;g/ 106 cells 
 Isotype control catalog number: IC108P</td>
 </tr>
 <tr>
 <td>Rat anti-mouse CCR2</td>
 <td>R&amp;D Systems</td>
 <td>FAB5538P</td>
 <td>Fluorophore conjugate: PE 
 Concentration: 0.1 &mu;g/ 106 cells 
 Isotype control catalog number: IC013P</td>
 </tr>
 <tr>
 <td>Armenian Hamster anti-mouse TCR&beta;</td>
 <td>BD Biosciences</td>
 <td>553174</td>
 <td>Fluorophore conjugate: APC 
 Concentration: 0.2 &mu;g/ 106 cells 
 Isotype control catalog number: 553956</td>
 </tr>
 <tr>
 <td>Rat anti-mouse CD8a</td>
 <td>BD Biosciences</td>
 <td>552877</td>
 <td>Fluorophore conjugate: PE-Cy7 
 Concentration: 0.8 &mu;g/ 106 cells 
 Isotype control catalog number: 552784</td>
 </tr>
 <tr>
 <td>Rat anti-mouse CD4</td>
 <td>BD Biosciences</td>
 <td>557956</td>
 <td>Fluorophore conjugate: Alexa Fluor 700 
 Concentration: 0.2 &mu;g/ 106 cells 
 Isotype control catalog number: 557963</td>
 </tr>
 </tbody>
</table>
Table 1. List of Materials and Reagents.

isolation of adipose tissue immune cells

La scoperta di una maggiore infiltrazione di macrofagi nel tessuto adiposo (AT) di roditori obesi e gli esseri umani ha portato ad un intensificarsi di interesse contributo delle cellule immunitarie all&#39;insulina locale e sistemica di resistenza. Isolamento e quantificazione delle diverse popolazioni di cellule immunitarie nel magri e obesi AT è oggi una tecnica comunemente utilizzata nei laboratori immunometabolism; ancora estrema cura deve essere presa sia in stromale isolamento delle cellule vascolari e nella analisi di citometria a flusso in modo che i dati ottenuti siano affidabili e interpretabile . In questo video mostriamo come per tritare, digerire, e isolare la frazione di cellule vascolari-arricchito stromale immune. Successivamente, mostriamo come anticorpi etichetta macrofagi e linfociti T e come correttamente cancello su di loro in esperimenti di citometria a flusso. Sono forniti di flusso rappresentativi citometria trame di basso contenuto di grassi alimentati magra e ricca di grassi nutriti topi obesi. Un elemento critico di questa analisi è l&#39;uso di anticorpi che fannoNon fluorescenza nei canali dove AT macrofagi sono naturalmente autofluorescenti, così come l&#39;uso di controlli di compensazione adeguati.

Collagenasi digestione di AT seguito da centrifugazione differenziale è stato utilizzato per isolare il SVF da cuscinetti di grasso dell&#39;epididimo di topi maschi C57BL/6J alimentato un basso contenuto di grassi (10% kcal dai grassi) o ad alto contenuto di grassi (60% kcal dai grassi) dieta (LFD e HFD , rispettivamente) per 16 settimane. Cellule del SVF furono poi etichettati con anticorpi primari fluoroforo-coniugato a quantificare la percentuale di vitale ATM (Figura 1) e AT cellule T (fig...

Watch this Scientific Journal Video about Isolation of Adipose Tissue Immune Cells at JoVE.com

Isolation of Adipose Tissue Immune Cells

Il tessuto adiposo (AT) è un sito di attivazione delle cellule immunitarie intensa e interazione. Quasi tutte le cellule del sistema immunitario sono presenti in AT e per i rapporti sono alterati da obesità. Isolamento adeguato, quantificazione e caratterizzazione di AT popolazioni di cellule immunitarie sono fondamentali per comprendere il loro ruolo nella malattia immunometabolic.

Isolamento di tessuto adiposo cellule immunitarie

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Research

JoVE Journal

Immunology and Infection

36.0K Views.  Vanderbilt University School of Medicine. Il tessuto adiposo (AT) è un sito di attivazione delle cellule immunitarie intensa e interazione. Quasi tutte le cellule del sistema immunitario sono presenti in AT e per i rapporti sono alterati da obesità. Isolamento adeguato, quantificazione e caratterizzazione di AT popolazioni di cellule immunitarie sono fondamentali per comprendere il loro ruolo nella malattia immunometabolic.

Video: Isolation of Adipose Tissue Immune Cells

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Misurazione dell&#39;attività Calpain nelle celle fissi e Living in citometria a flusso

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Ricerca

Immunologia e infezioni

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Sette passi per Stellate Cellule

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Analisi Citometria a flusso di cellule immunitarie all&#39;interno aorte Murine

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

Isolamento di cellule cardiache funzionali immunitario

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

Imaging intravitale del Timo mouse utilizzando 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Flusso Isolamento citometrica di primari murini di tipo II cellule alveolari epiteliali per gli studi funzionali e molecolari

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Isolamento e analisi citofluorimetrica delle cellule immunitarie dal cervello ischemica mouse

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

Isolamento, caratterizzazione e esame funzionale della cella di rete gengivale immunitario

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Reliable and High Efficiency Extraction of Kidney Immune Cells

Immune system activation occurs in multiple kidney diseases and pathophysiological processes. The immune system consists of both adaptive and innate components and multiple cell types. Sometimes, the cell type of interest is present in very low numbers among the large numbers of total cells isolated from the kidney. Hence, reliable and efficient isolation of kidney mononuclear cell populations is important in order to study the immunological problems associated with kidney diseases. Traditionally, tissue isolation of kidney mononuclear cells have been performed via enzymatic digestions using different varieties and strengths of collagenases/DNAses yielding varying numbers of viable immune cells. Recently, with the development of the mechanical tissue disruptors for single cell isolation, the collagenase digestion step is avoided and replaced by a simple mechanical disruption of the kidneys after extraction from the mouse. Herein, we demonstrate a simple yet efficient method for the isolation of kidney mononuclear cells for every day immune cell extractions. We further demonstrate an example of subset analysis of immune cells in the kidney. Importantly, this technique can be adapted to other soft and non-fibrous tissues such as the liver and brain.

Techniques that are reliable and efficient for the isolation of kidney immune cells are needed for downstream applications. This requires surface antibody labeling of a small number of kidney immune cells. Herein, we describe a concise method for isolation of kidney immune cells that seemingly achieves this goal.

Affidabile e di alta efficienza di estrazione del rene cellule immunitarie

Immune system activation occurs in multiple kidney diseases and pathophysiological processes.  The immune system consists of both adaptive and innate ...

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease.
This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) &#62;35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; agonist-rosiglitazone.
This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

Isolamento, espansione e adipogenico induzione di CD34 + CD31 + cellule endoteliali dal tessuto adiposo umano omentale e sottocutaneo

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...

Isolamento di tessuto adiposo cellule immunitarie

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Misurazione dell'attività Calpain nelle celle fissi e Living in citometria a flusso