Gli autori sono grati a Lisa Hansen e Kirsten Vestergaard per l'eccellente assistenza tecnica, e a P. Scherer, N. Joffin e C. Crewe per la lettura critica del manoscritto. Gli autori ringraziano l'UTSW Flow Cytometry Core per l'eccellente guida e assistenza nello sviluppo dei protocolli qui descritti. R.K.G. è supportato da NIH NIDDK R01 DK104789, NIDDK RC2 DK118620 e NIDDK R01 DK119163. J.P. è sponsorizzato da un premio pre-dottorato del Fondo per l'innovazione della Danimarca.

Tutti i protocolli e le procedure per gli animali sono stati approvati dal Comitato istituzionale per l'uso e la cura degli animali del Southwestern Medical Center dell'Università del Texas.
1. Isolamento della frazione vascolare stromale (SVF) dal tessuto adiposo bianco gonadico
<ol><li>Sezionare il tessuto adiposo bianco gonadico da topi di 6-8 settimane e posizionare i cuscinetti adiposi in 1x soluzione PBS.</li>
	<li>Combinare fino a 4 depositi di grasso (consigliati 2-4 depositi da 1-2 topi) e tritare il tessuto in un becher da 10 ml contenente 200 μl di tampone di digestione (1x HBSS, 1,5% BSA e 1 mg/mL di collagenasi D).</li>
	<li>Trasferire il tessuto macinato in una provetta da centrifuga da 50 ml contenente 10 ml di tampone per la digestione.</li>
	<li>Incubare la miscela a 37 °C a bagnomaria per 1 ora agitando.</li>
	<li>Filtrare il tessuto digerito attraverso un colino cellulare da 100 μm per rimuovere il tessuto non digerito.</li>
	<li>Diluire il campione filtrato a 30 mL con PBS contenente il 2% di FBS e centrifugare a 600 x g per 5 minuti a 4 °C. Aspirare il surnatante, che contiene adipociti maturi.</li>
	<li>Procedere immediatamente al metodo di isolamento cellulare preferito.</li>
</ol>2. Isolamento di APC e FIP mediante FACS
<ol><li>Sciogliere il pellet in 1 mL di tampone di lisi 1x RBC agitando delicatamente la provetta.</li>
	<li>Incubare a RT per 1-2 min.</li>
	<li>Aggiungere 10 mL di FBS/PBS al 2% e passare attraverso un filtro cellulare da 40 μm in una provetta da centrifuga pulita da 50 mL.</li>
	<li>Centrifugare a 600 x g per 5 min a 4 °C.</li>
	<li>Aspirare il terreno e risospendere il pellet in 400-800 μl di FBS/PBS al 2% contenente blocco Fc (1:200).</li>
	<li>Incubare a 4 °C per 10 min.</li>
	<li>Trasferire 400 μL-800 μL di sospensioni cellulari contenenti ≤106 cellule per mL in provette da 1,5 mL e aggiungere anticorpi. Le concentrazioni di anticorpi sono elencate nella sezione Tabella dei materiali .<ol><li>Preparare provette di controllo separate per 1) cellule non colorate, 2) controlli a colore singolo e 3) controlli FMO (fluorescenza meno uno).</li>
		<li>Colorare i campioni con gli anticorpi PDGFRβ, CD45, CD31, CD9, LY6C.</li>
	</ol></li>
	<li>Incubare a 4 °C per 15 min al riparo dalla luce.</li>
	<li>Centrifugare a 600 x g per 5 min a 4 °C.</li>
	<li>Aspirare i terreni e risospendere le cellule in 400 μl di FBS/PBS al 2%.</li>
	<li>Centrifugare a 600 x g per 5 min a 4 °C.</li>
	<li>Aspirare i terreni e risospendere le cellule in 400-800 μL di FBS/PBS al 2%. Quindi passare attraverso i tappi del filtro da 40 μm in provette a fondo tondo in polistirene da 5 mL per FACS.</li>
	<li>Segui i seguenti passaggi per il gating.<ol><li>Utilizzare controlli non colorati e monocolore per la compensazione.</li>
		<li>Usa i controlli FMO per impostare i gate sperimentali.</li>
		<li>Utilizzare la strategia di gating illustrata nella Figura 1 per ottenere APC e FIP. Gate APCs come CD45- CD31- PDGFRβ+ LY6C- CD9- e FIP come cellule CD45- CD31- PDGFRβ+ LY6C+ .</li>
	</ol></li>
	<li>Raccogliere le cellule selezionate in provette contenenti 500 μl di siero al 100% e mantenere le cellule in ghiaccio.</li>
	<li>Centrifugare le celle a 600 x g per 5 min a 4 °C. Aggiungere il 2% di FBS/PBS per lavare le celle centrifugando nuovamente a 600 x g per 5 minuti a 4 °C. Scartare il surnatante utilizzando le pile pellettate.</li>
	<li>Risospendere le cellule pellettate nel volume appropriato di terreno di coltura APC gonadico (per coltura cellulare) o tampone appropriato per l'analisi successiva.</li>
</ol>3. Separazione immunomagnetica delle frazioni adipogeniche e non adipogeniche
<ol><li>Isolare la SVF dal tessuto adiposo bianco gonadico seguendo i passaggi 1.1-1.7.</li>
	<li>Aspirare il surnatante e risospendere il pellet SVF in 10 mL di 1x tampone MS disponibile in commercio.</li>
	<li>Filtrare la sospensione cellulare attraverso un filtro cellulare da 40 μm.</li>
	<li>Centrifugare a 600 x g per 5 min a 4 °C.</li>
	<li>Aspirare il surnatante e risospendere le cellule pellettate in 100 μl di 1x MS Buffer.</li>
	<li>Eseguire la deplezione delle cellule CD31+ e CD45+ come discusso di seguito (Figura 2A, Passaggio 1). Questo viene fatto per rimuovere le cellule del lignaggio endoteliale ed ematopoietico.<ol><li>Contare le cellule utilizzando un contatore di cellule e regolare la concentrazione a ≤ 1 x 108 cellule/mL.</li>
		<li>Aggiungere gli anticorpi CD31 e CD45 coniugati con biotina alla sospensione cellulare, ciascuno a una concentrazione di ≤ 0,25 μg per 106 cellule e incubare su ghiaccio per 15 minuti.</li>
		<li>Lavare le celle aggiungendo 4 mL di 1x MS Buffer.</li>
		<li>Centrifugare a 600 x g per 5 min a 4 °C.</li>
		<li>Aspirare il surnatante e risospendere il pellet con 100 μL di tampone 1x MS.</li>
		<li>Aggiungere 10 μL di nanosfere di streptavidina e incubare le cellule su ghiaccio per 15 minuti.</li>
		<li>Lavare le celle aggiungendo 4 mL di tampone 1x MS.</li>
		<li>Centrifugare a 600 x g per 5 minuti a 4 °C, quindi aspirare il surnatante.</li>
		<li>Risospendere le cellule in 2,5 mL di 1 tampone MS e trasferirle in una provetta in polipropilene da 5 mL.</li>
		<li>Posizionare il tubo nella rastrelliera magnetica per 5 minuti.</li>
		<li>Raccogliere le cellule non marcate versando la sospensione cellulare in una provetta da centrifuga pulita da 15 mL. NOTA: Evitare di scuotere o asciugare le goccioline pendenti in quanto ciò porta alla contaminazione da frazione cellulare indesiderata.</li>
		<li>Rimuovere il tubo dal magnete e ripetere i passaggi 3.6.9. al punto 3.6.11. altre due volte per un totale di 3 lavaggi.</li>
	</ol></li>
	<li>Separazione delle frazioni adipogeniche e non adipogeniche (Figura 2A, Fase 2)<ol><li>Continua a lavorare con la frazione non etichettata (cioè la frazione CD45- CD31- ).</li>
		<li>Centrifugare la provetta da 15 mL contenente cellule non marcate a 600 x g per 5 minuti a 4 °C.</li>
		<li>Aspirare il surnatante e risospendere il pellet in 100 μl di tampone 1x MS.</li>
		<li>Aggiungere rispettivamente gli anticorpi LY6C coniugati con biotina e CD9 a una concentrazione di ≤ 0,25 μg per 106 cellule e incubare su ghiaccio per 15 minuti.</li>
		<li>Seguire i passaggi 3.6.3. al punto 3.6.12. per completare la seconda separazione.</li>
		<li>Raccogli le frazioni non legate. Le cellule non marcate (LY6C- CD9-) rappresentano la frazione adipogenica contenente APC (Figura 2A, Fase 3).</li>
		<li>Rimuovere la provetta dal magnete, risospendere ed eluire le cellule legate in 1x tampone MS. Gli eluati o frazione marcata (LY6C+ CD9+), rappresentano la frazione non adipogenica contenente FIP (Figura 2A, Fase 3).</li>
		<li>Centrifugare frazioni marcate e non marcate a 600 x g per 5 minuti in celle a pellet a 4 °C.</li>
	</ol></li>
	<li>Procedere immediatamente alla coltura cellulare (fase 6) o utilizzare precursori indifferenziati per le analisi preferite (fase 4 e/o fase 5).</li>
</ol>4. Valutare la purezza delle frazioni adipogeniche e non adipogeniche mediante citometria a flusso
<ol><li>Risospendere frazioni cellulari isolate da microsfere in 200-400 μL di blocco FBS/PBS al 2% contenente Fc (1:200).</li>
	<li>Incubare a 4 °C per 10 min.</li>
	<li>Trasferire la sospensione cellulare in provette da 1,5 mL e aggiungere gli anticorpi. Le concentrazioni di anticorpi sono elencate nella Tabella dei materiali.<ol><li>Preparare provette di controllo separate per 1) cellule non colorate, 2) controlli a colore singolo e 3) controlli FMO.</li>
		<li>Ai campioni, aggiungere gli anticorpi CD45, CD31, CD9 e LY6C.</li>
	</ol></li>
	<li>Incubare a 4 °C per 15 min al riparo dalla luce.</li>
	<li>Centrifugare a 600 x g per 5 minuti a 4 °C.</li>
	<li>Aspirare il surnatante e risospendere le cellule in 400 μL di FBS/PBS al 2%.</li>
	<li>Centrifugare la sospensione a 600 x g per 5 minuti a 4 °C.</li>
	<li>Aspirare il surnatante e risospendere le cellule in 400-800 μL di FBS/PBS al 2%. Filtrare attraverso tappi filtranti da 40 μm in provette a fondo tondo in polistirene da 5 mL per l'analisi mediante citometria a flusso.</li>
	<li>Analisi di citometria a flusso per valutare la purezza<ol><li>Utilizzare controlli non colorati e monocolore per la compensazione.</li>
		<li>Usa i controlli FMO per impostare i gate sperimentali.</li>
		<li>Utilizzare la strategia di gating per celle vive e singole come mostrato nella Figura 2B.</li>
		<li>Eseguire il gating come mostrato nella Figura 2B per CD45, CD31, LY6C e CD9.</li>
		<li>Utilizzare il software di citometria a flusso per valutare le frequenze delle cellule CD45, CD31, LY6C, CD9 (APC) e delle cellule CD45, CD31, LY6C+ (FIP) all'interno di frazioni isolate.</li>
	</ol></li>
</ol>5. Analisi dell'espressione genica mediante PCR quantitativa per valutare la purezza di FIP e APC
<ol><li>Estrarre l'mRNA da cellule isolate magneticamente o FACS utilizzando il kit di estrazione disponibile in commercio (vedere la Tabella dei materiali) seguendo le istruzioni del produttore.</li>
	<li>Utilizzare 1 μg di RNA per sintetizzare il cDNA utilizzando un kit per la trascrittasi inversa del cDNA (vedere la Tabella dei materiali) seguendo le istruzioni del produttore.</li>
	<li>Determinare i livelli relativi di mRNA dei geni selettivi per APC e FIP (Tabella 1) mediante PCR quantitativa utilizzando il master mix SYBR green PCR.<ol><li>Impostare la reazione del campione per la PCR come segue: 5 μL di 2x colorante verde SYBR, 1 μL di cDNA (~50 ng/μL), 0,5 μL di ciascun primer diretto e inverso (10 μM) e 3 μL di acqua.</li>
		<li>Utilizzare le seguenti condizioni standard di PCR per l'esecuzione quantitativa della PCR: fase di mantenimento: 50 °C per 2 min, 95 °C per 10 min; Fase PCR (40 cicli): 95 °C per 15 s, 60 °C per 1 min.</li>
	</ol></li>
	<li>Normalizzare i valori ai livelli del gene house-keeping (Rps18) calcolando ΔΔ-Ct.</li>
	<li>Per valutare la significatività statistica, eseguire il test t di Student non accoppiato.</li>
</ol>6. Colture cellulari e differenziamento
<ol><li>Centrifugare magneticamente o celle isolate FACS a 600 x g per 5 minuti a 4 °C.</li>
	<li>Risospendere il pellet in 500 μL di terreno di coltura APC gonadico e piastra 40K cellule/pozzetto da ciascuna frazione in una piastra di coltura a 48 pozzetti.</li>
	<li>Sostituire il supporto ogni 1-2 giorni. Le APC inizieranno a differenziarsi in adipociti man mano che si avvicinano alla confluenza. Le FIP mantenute nello stesso terreno sono resistenti all'adipogenesi.</li>
</ol>

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T.A.P. è un dipendente e azionista di Novo Nordisk A/S

Il ceppo di topi C57BL/6 è il ceppo di topo più utilizzato negli studi sull'obesità indotta dalla dieta. I topi C57BL/6 aumentano rapidamente di peso quando vengono sottoposti a una dieta ricca di grassi (HFD) e sviluppano alcune delle caratteristiche principali della sindrome metabolica associata all'obesità (ad esempio, insulino-resistenza e iperlipidemia). In particolare, l'espansione del WAT che si verifica in associazione con l'alimentazione con dieta ricca di grassi (HFD) si verifica in modo specifico del depos...

Il tessuto adiposo bianco (WAT) rappresenta il sito principale per l'accumulo di energia nei mammiferi. All'interno di questo tessuto, gli adipociti, o "cellule adipose", immagazzinano le calorie in eccesso sotto forma di trigliceridi, impacchettati in grandi goccioline lipidiche uniloculare. Inoltre, gli adipociti secernono una moltitudine di fattori che regolano vari aspetti dell'omeostasi energetica 1,2,3. Gli adipociti costituiscono la maggior parte del volume di WAT; tuttavia, gli adipociti rappresentano solo meno del 50% del totale delle cellule presenti in WAT 4,5. Il compartimento non adipocitario della WAT, o frazione stromale-vascolare (SVF), è piuttosto eterogeneo e contiene cellule endoteliali vascolari, cellule immunitarie residenti nei tessuti, fibroblasti e popolazioni di cellule precursori degli adipociti (APC).
WAT è eccezionale nella sua notevole capacità di espandersi in termini di dimensioni con l'aumento della domanda di accumulo di energia. Il mantenimento di questa plasticità tissutale è essenziale in quanto un'adeguata conservazione dei lipidi nel WAT protegge dalla deposizione deleteria di lipidi ectopici nei tessuti non adiposi6. Il modo in cui i singoli depositi WAT subiscono questa espansione in risposta all'eccesso calorico è un determinante critico della sensibilità all'insulina nel contesto dell'obesità7. L'espansione patologica del WAT, osservata in individui obesi con sindrome metabolica, è caratterizzata da un'espansione preferenziale dei depositi viscerali di WAT a scapito del tessuto adiposo sottocutaneo metabolicamente favorevole. Inoltre, l'insulino-resistenza nell'obesità è associata al rimodellamento patologico del WAT. Questo è caratterizzato da crescita ipertrofica degli adipociti esistenti (aumento delle dimensioni), angiogenesi inadeguata, infiammazione metabolica cronica, accumulo di componenti della matrice extracellulare (fibrosi) e ipossia tissutale 8,9. Questi fenotipi WAT dell'obesità sono associati alla steatosi epatica e all'insulino-resistenza, in modo simile a quanto osservato nella condizione di lipodistrofia (assenza di WAT funzionale). Al contrario, l'espansione sana del WAT si osserva nella popolazione obesa metabolicamente sana ed è caratterizzata da un'espansione preferenziale del WAT sottocutaneo protettivo e dall'espansione del deposito attraverso l'iperplasia degli adipociti10. Il reclutamento di nuovi adipociti è mediato dalla differenziazione de novo degli adipociti dalle cellule precursori degli adipociti (APC) (definita "adipogenesi"). L'iperplasia degli adipociti coincide con gradi relativamente più bassi di fibrosi WAT e infiammazione metabolica 6,11. Una moltitudine di tipi di cellule all'interno del microambiente WAT influenza direttamente la salute e l'espandibilità del WAT nell'obesità12. Pertanto, la definizione della funzione dei vari tipi di cellule presenti nel WAT rimane una priorità assoluta per il campo.
Nell'ultimo decennio, sono state impiegate diverse strategie per definire e isolare APC native da WAT SVF13 umano e murino. Tali strategie isolano le APC in base all'espressione sulla superficie cellulare di comuni marcatori di cellule mesenchimali/cellule progenitrici utilizzando tecniche di separazione cellulare basate su anticorpi. Questi approcci includono la selezione cellulare attivata dalla fluorescenza (FACS), utilizzando anticorpi marcati con fluorofori o la separazione immunomagnetica delle microsfere (cioè anticorpi chimicamente modificati). Le proteine della superficie cellulare bersaglio per l'isolamento delle APC includono PDGFRα, PDGFRβ, CD34 e SCA-1. Questi approcci hanno contribuito ad arricchire le APC; Tuttavia, le popolazioni cellulari isolate sulla base di questi marcatori sono piuttosto eterogenee. Recentissimi studi di sequenziamento dell'RNA a singola cellula (scRNA-seq) hanno evidenziato l'eterogeneità molecolare e funzionale delle cellule stromali all'interno della frazione stromale-vascolare isolata (SVF) del WAT murino 14,15,16,17. Dalle nostre analisi funzionali e scRNA-seq, abbiamo identificato e caratterizzato sottopopolazioni di cellule perivascolari PDGFRβ+ adipogeniche immunomodulanti e adipogeniche funzionalmente distinte nel compartimento stromale del WAT intra-addominale in topi adulti15. I precursori fibro-infiammatori, o FIP, rappresentano una sottopopolazione importante di cellule PDGFRβ+ e possono essere isolati in base all'espressione di LY6C (cellule LY6C+ PDGFRβ+)15. Le FIP mancano di capacità adipogenica, esercitano una forte risposta pro-infiammatoria a vari stimoli, producono collagene e secernono fattori anti-adipogenici15. L'attività pro-infiammatoria e fibrogenica di queste cellule aumenta in associazione con l'obesità nei topi, implicando queste cellule come regolatori del rimodellamento del WAT. La sottopopolazione LY6C- CD9- PDGFRβ+ rappresenta le cellule precursori degli adipociti (APC). Queste APC sono arricchite nell'espressione di Pparg e di altri geni pro-adipogenici e si differenziano facilmente in adipociti maturi in vitro e in vivo15. Qui, forniamo un protocollo dettagliato per l'isolamento di queste distinte popolazioni cellulari da depositi WAT intra-addominali di topi adulti utilizzando FACS e separazione immunomagnetica delle microsfere con anticorpi biotinilati. Questo protocollo può essere utilizzato per isolare sottopopolazioni progenitrici adipose funzionalmente distinte da più depositi WAT intra-addominali di topi adulti maschi e femmine15. Lo studio di queste popolazioni cellulari funzionalmente distinte in isolamento può contribuire notevolmente alla nostra attuale comprensione dei meccanismi molecolari che regolano l'adipogenesi e il rimodellamento del tessuto adiposo intra-addominale in salute e malattia.
Il protocollo seguente descrive in dettaglio l'isolamento dei progenitori adiposi dal WAT dell'epididimo murino; tuttavia, la stessa procedura può essere utilizzata per isolare le cellule corrispondenti dai depositi WAT mesenterico e retroperitoneale di topi maschi e femmine15. Un protocollo dettagliato su come identificare e isolare questi depositi nei topi può essere trovato in Bagchi et al.18. Questo protocollo è stato ottimizzato per l'uso di topi di 6-8 settimane di età. La frequenza e la capacità di differenziazione delle APC possono diminuire in associazione con l'invecchiamento.

<table>
	<tbody>
		<tr>
			<td>Mechanical Tissue Preparation and SVF Isolation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>40 and 100 &#181;m cell strainers</td>
			<td>Fisher Scientific</td>
			<td>352340/352360</td>
			<td></td>
		</tr>
		<tr>
			<td>1X Phosphate buffered saline (PBS)</td>
			<td>Fisher Scientific</td>
			<td>21040CV</td>
			<td></td>
		</tr>
		<tr>
			<td>5ml polypropylene tubes</td>
			<td>Fisher Scientific</td>
			<td>352053</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Digestion Buffer (for 10mL)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml HBSS</td>
			<td>Sigma</td>
			<td>H8264</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mg Collagenase D (1 mg/ml final cc.)</td>
			<td>Roche</td>
			<td>11088882001</td>
			<td></td>
		</tr>
		<tr>
			<td>0.15 g BSA (1.5 % final cc.)</td>
			<td>Fisher Scientific</td>
			<td>BP1605-100</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Immunomagnetic separation of APCs and non-APCs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5X MojoSort Buffer (MS buffer)</td>
			<td>BioLegend</td>
			<td>480017</td>
			<td></td>
		</tr>
		<tr>
			<td>5 ml MojoSort Magnet (MS magnet)</td>
			<td>BioLegend</td>
			<td>480019</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#181;L MojoSort Streptavidin Nanobeads</td>
			<td>BioLegend</td>
			<td>480015</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Purity Check and FACS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10X Red Blood Cell Lysis Buffer</td>
			<td>eBioscience</td>
			<td>00-4300-54</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc block (Mouse CD16/CD32)</td>
			<td>eBioscience</td>
			<td>553141</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin CD45</td>
			<td>BioLegend</td>
			<td>103103</td>
			<td>Concentration: &#8804; 0.25 &#181;g per 10^6 cells 
			Species: Mouse 
			Clone: 30-F11</td>
		</tr>
		<tr>
			<td>Biotin CD31</td>
			<td>BioLegend</td>
			<td>102503</td>
			<td>Concentration: &#8804; 0.25 &#181;g per 10^6 cells 
			Species: Mouse 
			Clone: MEC13.3</td>
		</tr>
		<tr>
			<td>Biotin CD9</td>
			<td>BioLegend</td>
			<td>124803</td>
			<td>Concentration: &#8804; 0.25 &#181;g per 10^6 cells 
			Species: Mouse 
			Clone: MZ3</td>
		</tr>
		<tr>
			<td>Biotin LY6C</td>
			<td>BioLegend</td>
			<td>128003</td>
			<td>Concentration: &#8804; 0.25 &#181;g per 10^6 cells 
			Species: Mouse 
			Clone: HK1.4</td>
		</tr>
		<tr>
			<td>CD31-PerCP/Cy5.5</td>
			<td>BioLegend</td>
			<td>102419</td>
			<td>Concentration: Dilution 1:400 
			Species: Mouse 
			Clone: 390</td>
		</tr>
		<tr>
			<td>CD45-PerCP/Cy5.5</td>
			<td>BioLegend</td>
			<td>103131</td>
			<td>Concentration: Dilution 1:400 
			Species: Mouse 
			Clone: 30-F11</td>
		</tr>
		<tr>
			<td>CD140b PDGFR&#946;-PE</td>
			<td>BioLegend</td>
			<td>136006</td>
			<td>Concentration: Dilution 1:50 
			Species: Mouse 
			Clone: APB5</td>
		</tr>
		<tr>
			<td>LY6C-APC</td>
			<td>BioLegend</td>
			<td>128016</td>
			<td>Concentration: Dilution 1:400 
			Species: Mouse 
			Clone: HK1.4</td>
		</tr>
		<tr>
			<td>CD9-FITC</td>
			<td>BioLegend</td>
			<td>124808</td>
			<td>Concentration: Dilution 1:400 
			Species: Mouse 
			Clone: MZ3</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture and Differentiation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gonadal APC Culture media (for 500mL)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>288 mL DMEM with 1 g/L glucose</td>
			<td>Corning</td>
			<td>10-014-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>192 mL MCDB201</td>
			<td>Sigma</td>
			<td>M6770</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Fetal bovine serum (FBS)** lot#14E024</td>
			<td>Sigma</td>
			<td>12303C</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL 100% ITS premix</td>
			<td>BD Bioscience</td>
			<td>354352</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL 10 mM L-ascorbic acid-2-2phosphate</td>
			<td>Sigma</td>
			<td>A8960-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>50 &#181;L 100 g/ml FGF-basic</td>
			<td>R&#38;D systems</td>
			<td>3139-FB-025/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Pen/Strep</td>
			<td>Corning</td>
			<td>30-001-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>500 &#181;L Gentamycin</td>
			<td>Gibco</td>
			<td>15750-060</td>
			<td></td>
		</tr>
		<tr>
			<td>**NOTE: The adipogenic capacity of primary APCs can vary from lot to lot of commercial FBS. Multiple lots/sources of FBS should be tested.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal adipose depots

La frazione stromale-vascolare (SVF) del tessuto adiposo bianco (WAT) è notevolmente eterogenea ed è costituita da numerosi tipi cellulari che contribuiscono funzionalmente all'espansione e al rimodellamento del WAT in età adulta. Un enorme ostacolo allo studio delle implicazioni di questa eterogeneità cellulare è l'incapacità di isolare facilmente sottopopolazioni cellulari funzionalmente distinte da WAT SVF per analisi in vitro e in vivo. La tecnologia di sequenziamento a singola cellula ha recentemente identificato sottopopolazioni di cellule perivascolari PDGFRβ+ fibro-infiammatorie e adipogeniche funzionalmente distinte in depositi di WAT intra-addominali di topi adulti. I progenitori fibro-infiammatori (denominati "FIP") sono cellule produttrici di collagene non adipogenico che possono esercitare un fenotipo pro-infiammatorio. Le cellule precursori degli adipociti (APC) PDGFRβ+ sono altamente adipogeniche sia in vitro che in vivo dopo il trapianto di cellule. Qui, descriviamo diversi metodi per l'isolamento di queste sottopopolazioni di cellule stromali da depositi murini di WAT intra-addominali. FIP e APC possono essere isolati mediante smistamento cellulare attivato da fluorescenza (FACS) o sfruttando la tecnologia delle microsfere immunomagnetiche basate su anticorpi biotinilati. Le cellule isolate possono essere utilizzate per l'analisi molecolare e funzionale. Lo studio delle proprietà funzionali della sottopopolazione di cellule stromali in isolamento amplierà le nostre attuali conoscenze sul rimodellamento del tessuto adiposo in condizioni fisiologiche o patologiche a livello cellulare.

Questo protocollo descrive due strategie che consentono l'isolamento di popolazioni di cellule stromali distinte da depositi WAT intra-addominali di topi adulti. Le APC e le FIP possono essere isolate mediante FACS (Figura 1) o separazione immunomagnetica delle microsfere con anticorpi biotinilati (Figura 2). Entrambi gli approcci utilizzano reagenti e anticorpi tutti disponibili in commercio. La separazione immunomagnetica delle microsfere porta alla separazion...

Read this Scientific Article Protocol about Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots at JoVE.com

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

Questo protocollo descrive l'approccio tecnico per isolare sottopopolazioni di cellule stromali adipogeniche e fibro-infiammatorie da depositi di tessuto adiposo bianco (WAT) murino intra-addominale mediante smistamento cellulare attivato dalla fluorescenza o separazione di microsfere immunomagnetiche.

Isolamento di sottopopolazioni di cellule stromali adipogeniche e fibro-infiammatorie da depositi adiposi intra-addominali murini

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute ...

isolation-adipogenic-fibro-inflammatory-stromal-cell-subpopulations

Research

JoVE Journal

Immunology and Infection

2.4K Views.  Novo Nordisk A/S. Questo protocollo descrive l'approccio tecnico per isolare sottopopolazioni di cellule stromali adipogeniche e fibro-infiammatorie da depositi di tessuto adiposo bianco (WAT) murino intra-addominale mediante smistamento cellulare attivato dalla fluorescenza o separazione di microsfere immunomagnetiche.

Video: Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

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 ...

Ricerca

Immunologia e infezioni

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-&#954;B (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-&#945; (TNF-&#945;), upon lipopolysaccharide (LPS) and Pam3CSK4 (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions.

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (Pam).

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

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Isolamento di cellule murine Linfonodo stromali

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

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

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Isolation, 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 ...

Simultaneous Study of the Recruitment of Monocyte Subpopulations Under Flow In Vitro

The recruitment of monocytes from the blood to targeted peripheral tissues is critical to the inflammatory process during tissue injury, tumor development and autoimmune diseases. This is facilitated through a process of capture from free flow onto the luminal surface of activated endothelial cells, followed by their adhesion and transendothelial migration (transmigration) into the underlying affected tissue. However, the mechanisms that support the preferential and context-dependent recruitment of monocyte subpopulations are still not fully understood. Therefore, we have developed a method that allows the recruitment of different monocyte subpopulations to be simultaneously visualized and measured under flow. This method, based on time-lapse confocal imaging, allows for the unambiguous distinction between adherent and transmigrated monocytes. Here, we describe how this method can be used to simultaneously study the recruitment cascade of pro-angiogenic and non-angiogenic monocytes in vitro. Furthermore, this method can be extended to study the different steps of recruitment of up to three monocyte populations.

Here, we present an integrated protocol that measures monocyte subpopulation trafficking under flow in vitro by use of specific surface markers and confocal fluorescence microscopy. This protocol can be used to explore sequential recruitment steps as well as to profile other leukocyte subtypes using other specific surface markers.

Studio simultaneo del reclutamento di sottopopolazioni monocito sotto flusso In Vitro

The recruitment of monocytes from the blood to targeted peripheral tissues is critical to the inflammatory process during tissue injury, tumor development ...

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected in the urinary tract. The protocol can be broadly divided into three sections: the infection, bladder epithelial cell harvesting, and mouth micropipetting to isolate individual infected epithelial cells. The isolated epithelial cell contains viable bacterial cells and is nearly free of contaminating extracellular bacteria, making it ideal for downstream single-cell analysis. The time taken from the start of infection to obtaining a single intracellular bacterial community is about 8 h. This protocol is inexpensive to deploy and uses widely available materials, and we anticipate that it can also be utilized in other infection models to isolate single infected cells from cell mixtures even if those infected cells are rare. However, due to a potential risk in mouth micropipetting, this procedure is not recommended for highly infectious agents.

This protocol describes a simple method of isolating single, infected-bladder epithelial cells from a murine model of urinary tract infection.

Isolamento di singole comunità batteriche intracellulari generati da un modello murino di infezione delle vie urinarie per analisi unicellulare a valle

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected ...

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn&#8217;s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

Isolamento delle cellule mononucleari Di Lamina propria dal colon Murine utilizzando Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...

Isolation of Macrophage Subsets and Stromal Cells from Human and Mouse Myocardial Specimens

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative responses after cardiac injury. How various subsets of macrophages orchestrate the immune responses after cardiac injury is an active area of research. Presented here is a simple protocol that our lab performs routinely, for the extraction of macrophages from mouse and human myocardium specimens obtained from healthy and diseased individuals. Briefly, this protocol involves enzymatic digestion of cardiac tissue to generate a single cell suspension, followed by antibody staining, and flow cytometry. This technique is suitable for functional assays performed on sorted cells as well as bulk and single cell RNA sequencing. A major advantage of this protocol is its simplicity, minimal day to day variation and wide applicability allowing investigation of macrophage heterogeneity across various mouse models and human disease entities.

Presented here is a protocol to isolate various subsets of macrophages and other non-immune cells from human and mouse myocardium by preparing a single cell suspension through enzymatic digestion. Gating schemes for flow cytometry based identification and characterization of isolated macrophages are also presented.

Isolamento di sottoinsiemi di macrofaschi e cellule stromali da esemplari di miocardio umano e topo

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative ...

Isolating Brown Adipocytes from Murine Interscapular Brown Adipose Tissue for Gene and Protein Expression Analysis

Brown adipose tissue (BAT) is responsible for non-shivering thermogenesis in mammals, and brown adipocytes (BAs) are the functional units of BAT. BAs contain both multilocular lipid droplets and abundant mitochondria, and they express uncoupling protein 1 (UCP1). BAs are categorized into two sub-types based on their origin: embryo derived classical BAs (cBAs) and white adipocytes derived BAs. Due to their relatively low density, BAs cannot be isolated from BAT with traditional centrifugation method. In this study, a new method was developed to isolate BAs from mice for gene and protein expression analysis. In this protocol, interscapular BAT from adult mice was digested with Collagenase and Dispase solution, and the dissociated BAs were enriched with 6% iodixanol solution. Isolated BAs were then lysed with Trizol&#160;reagent for simultaneous isolation of RNA, DNA, and protein. After RNA isolation, the organic phase of the lysate was used for protein extraction. Our data showed that 6% iodixanol solution efficiently enriched BAs without interfering with follow-up gene and protein expression studies. Platelet-derived growth factor (PDGF) is a growth factor that regulates the growth and proliferation of mesenchymal cells. Compared to the brown adipose tissue, isolated BAs had significantly higher expression of Pdgfa. In summary, this new method provides a platform for studying the biology of brown adipocytes at a single cell-type level.

This study describes a new method of isolating murine brown adipocytes for gene and protein expression analysis.

Isolamento di adipociti bruni da tessuto adiposo bruno interscapolare murino per l'analisi dell'espressione genica e proteica

Brown adipose tissue (BAT) is responsible for non-shivering thermogenesis in mammals, and brown adipocytes (BAs) are the functional units of BAT. BAs ...