Gli autori riconoscono il supporto del Australian Health and Medical Research Council (progetto grant 1012409 collegato a BHC).

Questo protocollo è stato approvato dal sud orientale Sydney umana ricerca comitato etico e ratificato dal comitato di etica umana ricerca l'Università del New South Wales. Sangue del cordone ombelicale ottenuto da donatori sani è stato fornito da Sydney Cord Blood Bank (Sydney, NSW, Australia). Volumi di circa 100 mL sono stati utilizzati per questa procedura.
Nota: Lavorare in una classe II biosicurezza utilizzando una tecnica asettica. Decontaminare l'esterno della sacca del sangue con etanolo al 70%. Per questa procedura, usare strumenti sterili (forbici, pinzette).
1. cavo di preparazione delle cellule del sangue e l'isolamento di CD34 cellule+
<ol>
	<li>Preparare il tampone di separazione sterile (SB) con tamponato fosfato salino (PBS), a pH 7,2 e contenente 0.5% bovino dell'albumina di siero e 2 mM EDTA.</li>
	<li>Versare 10 mL di SB in una provetta conica da 50 mL (una provetta per 10 mL di sangue è necessaria). Utilizzando un ago smussato G18 montato su una siringa da 10 mL, disegnare 10 mL di sangue dal sacchetto e dispensare nelle provette da 50 mL contenente 10 mL di SB.</li>
	<li>Aggiungere 15 mL di media di separazione del linfocita (Vedi Tabella materiali) alla parte inferiore del tubo contenente il sangue diluito, creando due strati. Nota: Dispensare media lentamente nella parte inferiore del tubo per evitare di miscelare i diversi strati.</li>
	<li>Centrifugare provette a 1.200 × g per 30 min a temperatura ambiente (TA) con senza pausa e senza accelerazione.</li>
	<li>Trasferire lo strato vicino al centro del tubo contenente cellule mononucleari (circa 5-10 mL da ciascuna provetta) in una nuova provetta da 50 mL utilizzando una pipetta Pasteur. Aggiungere SB ogni tubo per un volume totale di 50 mL.</li>
	<li>Centrifugare a 400 × g per 10 min a RT. scartare il surnatante.</li>
	<li>Risospendere il pellet cellulare accuratamente con una pipetta Pasteur in 5-10 mL di SB. Combine le cellule sospese e portare il volume a 50 mL con SB.</li>
	<li>Numero di celle utilizzando la macchiatura blu di Trypan e un emocitometro o automatizzata delle cellule contatore (Vedi Tabella materiali). Per determinare la percentuale di CD34+ cellule nel campione, risospendere 2,5 × 105 cellule in 100 µ l di SB e macchia aggiungendo 10 µ l di anticorpo anti-CD34-PE per 15-30 min a 4 ° C. Analizzare mediante citometria a flusso (Figura 1A).</li>
	<li>Provette per centrifuga a 400 × g per 10 min a 4 ° C. Gettare il surnatante. Nota: se non richiesto immediatamente, cellule mononucleari possono essere congelate in questa fase.</li>
	<li>Risospendere le cellule in 300 µ l di SB per 108 cellule. Aggiungere 100 µ l di IgG umane FcR (reagente, bloccante Vedi Tabella materiali) e 100 µ l di CD34 biglie magnetiche per 108 cellule. Mescolare delicatamente e incubare a 4 ° C per 30-40 min. Nota: Una sospensione singola cella è necessaria (se necessario, passare le cellule però un filtro 30 µm prima di aggiungere i reagenti). Utilizzare i volumi di reagente descritti qui per 108 celle o meno. Per più di 108 celle, scalabilità di conseguenza i reagenti (SB, bloccando la soluzione e sfere magnetiche CD34).</li>
	<li>Preparare la colonna di separazione LS durante il periodo di incubazione posizionando la colonna LS in un supporto magnetico e lavaggio con 3 mL di SB. Discard effluente. Nota: Colonne di separazione LS possono essere caricate con fino a 2 × 108 cellule totali. Colonne più piccole e più grandi sono disponibili.</li>
	<li>Aggiungere 5-10 mL di SB per la miscela di cella e centrifugare a 400 × g per 10 min. scartare il surnatante.</li>
	<li>Risospendere le cellule in 1,5 mL di SB e caricare la sospensione cellulare (1,5 mL) sulla colonna di LS. Raccogliere il flusso attraverso contenente le celle senza etichetta in un tubo di raccolta di 15 mL (frazione negativo #1). Lasciate che il liquido svuotare e lavare la colonna con 1,5 mL di SB.</li>
	<li>Caricare il flusso raccolto attraverso (3 mL) nuovamente dentro la colonna e raccogliere il flusso attraverso nello stesso tubo di raccolta 15 mL (frazione negativo #1). Lavare la colonna LS 3 volte con 3 mL di SB. Nota: Se necessario, le cellule nella frazione negativa (12 mL) possono essere macchiate con l'anticorpo anti-CD34 per accertare la cattura di CD34+ cellule tramite la colonna di LS.</li>
	<li>Togliere colonna dal separatore magnetico e lavare le cellule lentamente con lo stantuffo della siringa in una nuova provetta da 15 mL con 2 mL di SB.</li>
	<li>Riposizionare colonna sul separatore magnetico e carico da 2 mL di celle nella colonna. Raccogliere il flusso attraverso e lavare con 2ml SB. Questa è la frazione negativa #2 (4 mL).</li>
	<li>Rimuovere la colonna LS da separatore magnetico e aggiungere 2 mL di Flush SB. cellule costantemente e saldamente con un pistone della siringa per raccogliere il CD34+ frazione delle cellule. Contare le celle usando la macchiatura blu di trypan e un emocitometro o contatore di cellule automatizzato.</li>
	<li>Provetta da centrifuga con la frazione positiva a 400 × g per 15 min a 4 ° C. Scartare il surnatante. Risospendere le cellule in terreni privi di siero per CD34+ cellule (SFM, Vedi Tabella materiali). Nota: se non richiesto immediatamente, le cellule possono essere congelate in azoto liquido in questa fase.</li>
</ol>
2. purezza Check di CD34 isolato cellule+
<ol>
	<li>2 x 104 cellule dalla frazione positiva con 10 µ l l'anticorpo anti-CD34-PE e anticorpo anti-CD45-PerCP 20 µ l per 15 min a 4 ° C (uso un separato del tubo per i controlli dell'isotipo) (Figura 1B).</li>
	<li>Aggiungere 1 mL di SB per lavare. Centrifugare a 400 × g per 10 min. Risospendere il pellet in 200 µ l di SB.</li>
	<li>Eseguire l'analisi cytometric di flusso e cancello la popolazione di cellule vive per escludere detriti (Figura 1B). Impostare la porta per le popolazioni positivi PE e PerCP utilizzando i controlli di isotipo PE e PerCP (Figura 1B) e determinare la percentuale di CD34+/CD45+ cellule.</li>
</ol>
3. differenziamento megacariocitico
<ol>
	<li>Seme 5 × 105 cellule/mL CD34+ cellule in 2 mL di SFM completati con 50 ng/mL trombopoietina umana ricombinante (terapeutici) per pozzetto in un piatto di 12-pozzetti. Incubare le cellule a 37 ° C, 5% CO2 in atmosfera umidificata. Se le cellule sono confluenti prima che sono necessari per l'analisi, raccogliere le cellule e dividere in pozzi multipli con media fresco e terapeutici. Nota: Due o tre pozzetti devono essere preparati specificamente per controllare la differenziazione in diversi momenti (ad esempio, giorni 7, 9 e 10).</li>
	<li>Raccogliere le cellule dai pozzetti accantonati per controllare la differenziazione senza disturbare le cellule in altri pozzi.</li>
	<li>Cellule di macchia con l'anticorpo anti-GPIIb/CD41-FITC di 20 µ l e 10 µ l anti-GPIX/CD42a-Alexa Fluor 647 anticorpo in un volume finale di 100 µ l. impostare un controllo tubo usando gli anticorpi di controllo rispettivi isotype. Incubare per 15-30 min a 4 ° C.</li>
	<li>Aggiungere 1 mL di SB per lavare.</li></ol>Centrifugare a 400 × g per 10 min. scartare il supernatante e risospendere il pellet in 200-300 µ l di SB. analizzare tramite flusso cytometry.
	<li>Per ogni fluoroforo, analizzare i controlli di isotipo per impostare il gating per popolazioni positivi FITC e Alexa 647 di farina. Analizzare i campioni di cellule macchiato per determinare la percentuale di CD41+/CD42a+ doppio cellule positive, che rappresentano maturo MK (Figura 1).</li>
	<li>Per visualizzazione microscopica della cellula marcatori di superficie macchiano le celle come descritto al punto 3.3.<ol>
		<li>Aggiungere 1 mL di SB per lavare. Centrifuga a 400 × g per 10 min. Risospendere il pellet in 100 µ l di SB e spin su una lastra di vetro a 1.000 x g per 5 min. Fix cellule sul vetrino mediante immersione in metanolo per 30 s. aria secca, aggiungere 20 µ l di montaggio supporti contenenti DAPI (Vedi Tabella materiali) , coprire con un vetrino coprioggetto e visualizzare utilizzando un microscopio a fluorescenza (Figura 2A).</li>
	</ol>
	</li>
	<li>Per la visualizzazione di antigeni intracellulari, risospendere le cellule in PBS e fissate con paraformaldeide (1% di concentrazione finale) per 15 min a temperatura ambiente. Per permeabilize le celle, aggiungere triton-X100 (0,1%) e incubare per 15 min. cellule di lavaggio con 2 mL PBS/0.1% triton-X 100. Risospendere in 100 µ l di PBS/0.1% triton-X 100, aggiungere anti vWf e anti CD62p anticorpi (diluizione 1: 200) ed incubare per 30 min a temperatura ambiente.<ol>
		<li>Lavare con 2 mL PBS/0.1% triton-X100 e centrifugare a 400 × g per 10 min, risospendere in 100 µ l di buffer stesso e aggiungere anti-mouse IgG-Alexa 594 e anti-coniglio IgG-Alexa 488 (1: 100). Incubare per 30 min a temperatura ambiente, lavare con 2 mL PBS/0.1% triton-X100, risospendere in 100 µ l di buffer stesso e aggiungere 20 µ l di anti CD42b-APC. Poi girare le cellule su vetrini come descritto al punto 3.6.1 e preparare i campioni per visualizzazione microscopica come descritto al punto 3.6.1.</li>
	</ol>
	</li>
	<li>Per la determinazione di ploidia, raccogliere le cellule ai giorni 12 o 13 di differenziazione. Aggiungere 1 mL di SB per lavare. Centrifuga a 400 × g per 10 min e macchia con l'anticorpo anti-GPIIb/CD41-FITC di 20 µ l in un volume finale di 100 µ l. Incubare a 4 ° C per 30 min.<ol>
		<li>Lavare una volta con 1 mL di SB e risospendere il pellet in 300 µ l di citrato ipotonico buffer (citrato di sodio di 1,25 mM, 2,5 mM di cloruro di sodio, destrosio di 3,5 mM) contenenti 20 µ g/ml ioduro di propidio e 0,05% Triton-X100. Incubare per 15 min a 4 ° C al riparo dalla luce.</li>
		<li>Aggiungere RNasi a una concentrazione finale di 20 µ g/mL ed incubare per 30 min a 4 ° C al riparo dalla luce. Determinare l'intensità di ioduro di propidio tramite flusso cytometry raccogliendo 30.000 a 50.000 eventi della popolazione CD41-FITC+ (Figura 2).</li>
	</ol>
	</li>

4. a conteggio, enumerazione della piastrina e l'attivazione piastrinica
<ol>
	<li>Raccogliere le cellule (dal punto 3.1) a giorni 8 o 9 di differenziazione e di sementi a 1 × 104 cellule/pozzetto in piastre 48 pozzetti in 200 µ l di fresco SFM completati con 50 ng/mL terapeutici. Cultura per 5 giorni a 37 ° C, 5% CO2. Nota: Ai fini della quantificazione, seme pozzi in triplice copia. Questa bassa densità è necessaria per la visualizzazione e contiamo di a-cuscinetto Mk. associato solitamente iniziano ad apparire dopo 2 giorni di cultura. Il picco è tra i giorni 4 e 5.</li>
	<li>Contare il numero di a-cuscinetto MK nel pozzo tutto su un microscopio invertito luce utilizzando 10 X o 20 X obiettivi. Nota: Una fase di microscopio riscaldata (37 ° C) è preferibile, dato che la conservazione delle cellule a temperatura ambiente per periodi prolungati provoca restringimento delle estensioni a. Associato è osservati come lunghe estensioni dal corpo MK. Ogni MK può avere diversi a sporgenze. Come sviluppa associato, il corpo della MK diminuisce nel formato.</li>
	<li>Raccogliere le cellule e centrifugare a 400 x g per 10 min a temperatura ambiente. Macchia le cellule con l'anticorpo di anti-umani CD41-FITC 20 µ l, come descritto ai punti 3.3 e 3.4. Calcolare la percentuale di MK a cuscinetto (pbMK): pbMK (%) = [(MKs propiastrine-cuscinetto/bene) / (Totale CD41+ cellule / bene)] x 100</li>
	<li>Per contare le piastrine rilasciate nel terreno di coltura, delicatamente mescolare le cellule con una pipetta Pasteur e raccogliere 100 µ l a giorni 14 e 15 della cultura.<ol>
		<li>Macchiare con 20 µ l anticorpo anti-umane CD41-FITC per 20-30 min a 4 ° C. Impostare una provetta di controllo utilizzando l'anticorpo di controllo rispettivi isotype.</li>
		<li>Aggiungere 150 µ l di SB e 50 µ l di contare le perline.</li>
		<li>Per l'analisi cytometric di flusso, impostato il FSC e SSC scala logaritmica. Piastrine del sangue umane normale uso da plasma ricco di piastrine colorati con CD41-FITC come descritto in sezione 4.4.1 per impostare il gating per le piastrine (Figura 3A).</li>
		<li>Analizzare le piastrine macchiate con perline di conteggio tramite flusso cytometry. Raccogliere 1.000 eventi di conteggio perline utilizzando il FSC rispetto al grafico a dispersione SSC (Figura 3A). Calcolare il numero delle piastrine in base agli eventi positivi CD41-FITC (Figura 3B) utilizzando la formula: Piastrine per µ l = [(numero di eventi positivi CD41-FITC)/1,000 beads)] x [(number of beads in 50 µL)/volume del campione)]</li>
	</ol>
	</li>
	<li>Per analizzare l'attivazione della piastrina, delicatamente mescolare le cellule con una pipetta Pasteur e raccogliere 100 µ l a giorni 14 e 15 della cultura. Aggiungere 1 mL di tampone (137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1,8 mM CaCl2, 0,2 mM Na2HPO4, 12mm NaHCO3, 5,5 mM D-glucosio, pH 6.5) e centrifugare di Tyrode a 200 x g per 5 min agglomerare le cellule.<ol>
		<li>Raccogliere il surnatante e centrifuga a 800 x g per 10 min a particelle di dimensioni della piastrina di pellet.</li>
		<li>Eliminare il surnatante e risospendere in 100 µ l di tampone di Tyrode. Aggiungere 20 µ l di anticorpo PAC1-FITC e adenosina difosfato (ADP) ad una concentrazione finale di 20 µM. Incubare a temperatura ambiente per 20 min. analisi di citometria a flusso per determinare la percentuale di eventi positivi FITC. Uso delle piastrine umane fresche trattate nello stesso modo come controllo positivo (Figura 3).</li>
	</ol>
	</li>
</ol>

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I., Khachigian, L., Perdomo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug-induced+Immune+Thrombocytopenia.">Drug-induced Immune Thrombocytopenia.</a> Hematol Oncol Clin North Am. 27 (3), (2013).</li><li>Stasi, R., 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=Idiopathic+thrombocytopenic+purpura:+Current+concepts+in+pathophysiology+and+management.">Idiopathic thrombocytopenic purpura: Current concepts in pathophysiology and management.</a> Thromb Haemost. 99 (1), 4-13 (2008).</li></ol>

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Il protocollo descritto qui è adatto a produzione costante MK e piastrine in coltura dal sangue del cordone ombelicale. Queste cellule possono essere utilizzate per studiare i vari processi come l'effetto di droghe o di attività biologiche su MK proliferazione, differenziazione, a formazione e produzione della piastrina.
Una varietà di terreni di coltura e combinazioni di citochina sono stati presentati nella letteratura. Addizione di citochine come il fattore di cellula formativa, ligando ...

Isolamento di un numero adeguato di megacariociti umani primari (MK) per uso di laboratorio regolari non è fattibile a causa della loro bassa frequenza nel midollo osseo, dove rappresentano ~0.01% di cellule nucleate1. Una comoda alternativa è l'espansione ex vivo e la differenziazione delle staminali ematopoietiche e cellule progenitrici in presenza di specifici fattori di crescita. Una serie di citochine, compreso il fattore di cellula formativa (SCF; c-kit ligando) e interleuchina (IL) -3 e IL-11 è stati impiegati in sistemi di coltura per produrre MKs. Thrombopoietin (TPO) è il fattore più efficace di crescita e differenziazione megakaryocytic culture ed è efficace da solo o con altre citochine, quali SCF e IL-32. TPO può agire su popolazioni di cellule staminali per provocare la proliferazione e la maturazione di MKs2.
Le piastrine di produrre MK da protrusioni citoplasmatiche chiamati associato e, in vivo, circa 1 x 1011 piastrine sono formate ogni giorno per sostenere la conta piastrinica di 150-400 x 109produzione /L. piastrinica in vitro è fino a 1000 -piegare inferiori lo in vivo stima3, e ciò ha dato luogo a numerose condizioni di coltura utilizzando CD34+ cellule progenitrici ematopoietiche per migliorare della piastrina e MK produzione in vitro. La fonte iniziale di CD34+ celle utilizzate per il differenziamento di MK era sangue periferico umano4. Includono altre fonti di cellule del midollo osseo5,6, cellule staminali embrionali/induced pluripotent cellule staminali (ESC/iPSC)7e cordone ombelicale sangue (UCB)8,9,10 . Midollo osseo umano CD34+ 11 e mouse lignaggio negativo del midollo osseo cellule5 prodotti MK e piastrine in vitro; Tuttavia, la mancanza di disponibilità del midollo osseo umano limita il suo uso come fonte di CD34+ cellule. Al contrario, ESC e iPSC rappresentano una fonte illimitata di cellule per la produzione della piastrina in vitro . La produzione della piastrina da queste cellule richiede le cellule di alimentatore come cellule murine OP9 e periodi più lunghi di cultura. Le piastrine derivate in condizioni prive di alimentatore sembrano essere meno funzionale12. iPSC-derivato piastrine rischiano di essere di uso nelle regolazioni cliniche, dal momento che può essere espansa a larga scala. Questo processo richiede trasduzione lentivirale-mediata di fattori di trascrizione e a lungo termine delle cellule cultura13.
UCB è una fonte accessibile di CD34+ cellule che possono essere facilmente utilizzate in impostazioni di ricerca. TPO da solo può promuovere la differenziazione del midollo derivate dal sangue CD34+ cellule e questo dà luogo ad elevata purezza, maturo MKs senza l'esigenza del completamento del siero o co-coltura con le cellule di alimentatore. Altre citochine come SCF può diminuire la differenziazione da UCB CD34 cellule+ , mentre ligando Flt-3 e IL-11 promuovere la produzione di megacariociti acerbi14. Questo protocollo descrive la produzione di colture altamente pure MK cordonali CD34+ cellule nelle circostanze senza siero.

<table>
	<tbody>
		<tr>
			<td>Cell Culture Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human TPO</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-013</td>
			<td></td>
		</tr>
		<tr>
			<td>StemSpan SFEM II</td>
			<td>Stem Cell Technologies</td>
			<td>9605</td>
			<td>Serum-free media for CD34+ cells</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>CD34 Isolation Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CD34 MicroBead kit ultrapure</td>
			<td>Miltenyi Biotec</td>
			<td>130-100-453</td>
			<td>This kit includes the FcR human IgG blocking reagent and CD34 microbeads. These beads contain the anti-CD34 antibody clone QBEND/10. Use a different anti-CD34 clone for purity check (e.g. clone 8G12).</td>
		</tr>
		<tr>
			<td>Lymphoprep</td>
			<td>Alere Technologies</td>
			<td>1114545</td>
			<td>Lymphocyte separation media (density 1.077 g/mL)</td>
		</tr>
		<tr>
			<td>Sterile separation buffer (SB)</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-221</td>
			<td>This buffer contains phosphate buffered saline (PBS), pH 7.2 containing 0.5% bovine serum albumin and 2 mM EDTA. It can be prepared using sterile, cell culture grade components. De-gas before use because air bubbles can block the column.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Flow Cytometry and Cell Staining Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PE Mouse anti-Human CD34</td>
			<td>BD Biosciences</td>
			<td>340669</td>
			<td>Clone 8G12. This can be used for CD34 purity check. Final antibody concentration 1:10 dilution.</td>
		</tr>
		<tr>
			<td>PerCP mouse anti-human CD45</td>
			<td>BD Biosciences</td>
			<td>347464</td>
			<td>1:10 dilution</td>
		</tr>
		<tr>
			<td>PerCP isotype control</td>
			<td>BD Biosciences</td>
			<td>349044</td>
			<td>1:10 dilution</td>
		</tr>
		<tr>
			<td>FITC Mouse anti-Human CD41a</td>
			<td>BD Biosciences</td>
			<td>340929</td>
			<td>Final antibody concentration 1:5 dilution.</td>
		</tr>
		<tr>
			<td>APC Mouse anti-Human CD42b</td>
			<td>BD Biosciences</td>
			<td>551061</td>
			<td>This antibody can also be used to detect mature MK (the percentage of positive cells in usually lower than with anti CD42a). Final antibody concentration 1:10 dilution.</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 Mouse anti-Human CD42a</td>
			<td>AbD Serotec</td>
			<td>MCA1227A647T</td>
			<td>Currently distributed by Bio-Rad. Final antibody concentration 1:10 dilution.</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 Mouse Negative Control</td>
			<td>AbD Serotec</td>
			<td>MCA928A647</td>
			<td>Currently distributed by Bio-Rad. Isotype control antibody</td>
		</tr>
		<tr>
			<td>Anti von Willebrand factor rabbit polyclonal</td>
			<td>Abcam</td>
			<td>AB6994</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>V450 mouse anti-humna CD41a</td>
			<td>BD Biosciences</td>
			<td>58425</td>
			<td>1: 20 dilution</td>
		</tr>
		<tr>
			<td>V450 isotype control</td>
			<td>BD Biosciences</td>
			<td>580373</td>
			<td>1:20 dilution</td>
		</tr>
		<tr>
			<td>PAC1-FITC antibody</td>
			<td>BD Biosciences</td>
			<td>340507</td>
			<td>1:10 dilution</td>
		</tr>
		<tr>
			<td>Anti CD62p mouse monoclonal</td>
			<td>Abcam</td>
			<td>AB6632</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti rabbit IgG</td>
			<td>Invitrogen</td>
			<td>A11008</td>
			<td>1:100 dilution</td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 goat anti mouse IgG</td>
			<td>Invitrogen</td>
			<td>A11020</td>
			<td>1:100 dilution</td>
		</tr>
		<tr>
			<td>Ig Isotype Control cocktail-C</td>
			<td>BD Biosciences</td>
			<td>558659</td>
			<td>Isotype control antibody</td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Sigma Aldrich</td>
			<td>P4864</td>
			<td></td>
		</tr>
		<tr>
			<td>CountBright Absolute Counting Beads</td>
			<td>Molecular Probes, Invitrogen</td>
			<td>C36950</td>
			<td>Counting beads</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td>Smaller and larger columns are also commercially available</td>
		</tr>
		<tr>
			<td>MidiMACS Separator magnet</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-302</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS MultiStand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 5mL round bottom polypropylene FACS tubes, with Snap Cap, Sterile</td>
			<td>In Vitro technologies</td>
			<td>352063</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Menzel-Glaser</td>
			<td>J3800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting media with DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td>Antifade mounting medium with DAPI</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Leica</td>
			<td>DMIRB inverted microscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Zeiss</td>
			<td>Vert.A1</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell analyser</td>
			<td>BD Biosciences</td>
			<td>FACS Canto II</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytospin centrifuge</td>
			<td>ThermoScientific</td>
			<td>Cytospin 4</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell analyser software</td>
			<td>BD Biosciences</td>
			<td>FACS Diva Software</td>
			<td></td>
		</tr>
		<tr>
			<td>Single cell analysis software</td>
			<td>Tree Star</td>
			<td>FlowJo</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscope software</td>
			<td>Zeiss</td>
			<td>Zen 2 blue edition</td>
			<td></td>
		</tr>
	</tbody>
</table>

megakaryocyte differentiation and platelet formation from human cord blood-derived cd34+ cells

La produzione della piastrina si verifica principalmente nel midollo osseo in un processo noto come thrombopoiesis. Durante thrombopoiesis, cellule progenitrici ematopoietiche differenziano ai precursori delle piastrine forma chiamati megacariociti, che differenziano terminalmente per rilasciare le piastrine da processi citoplasmici lungo definiti associato. I megacariociti sono confinate al midollo osseo le cellule rare e sono quindi difficili da raccogliere in numero sufficiente per uso di laboratorio. Produzione efficiente di megacariociti umani può essere raggiunto in vitro coltivando CD34+ cellule nelle circostanze adatte. Il protocollo dettagliato qui descrive l'isolamento di CD34 cellule di+ da magnetico cella ordinamento da campioni di sangue del cordone ombelicale. Sono descritti i passi necessari per la produzione di megacariociti altamente puri, maturi nelle circostanze senza siero. Dettagli di analisi fenotipica di differenziamento megacariocitico e determinazione della produzione della piastrina e di formazione a disposizione. Gli effettori che influenzano la differenziazione dei megacariociti e/o a formazione, quali gli anticorpi anti-piastrine o mimetics della trombopoietina, possono essere aggiunti alle cellule coltivate per esaminare la funzione biologica.

Questo protocollo permette la preparazione di colture di MK altamente pure dal cordone derivate dal sangue CD34+ cellule. La percentuale di CD34+ cellule nel midollo sangue è di circa 1,3%15 (Figura 1A) e il numero totale di cellule mononucleari (passo 1,8) intervalli da 90-300 x 106 per unità UCB. La purezza di CD34 + cellule CD45 + dopo intervalli di isolamento da 90 a 99% (Figu...

Watch this Scientific Journal Video about Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells at JoVE.com

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells

Una popolazione altamente pura dei megacariociti sono ottenibili dal midollo derivate dal sangue CD34+ cellule. Un metodo per CD34+ differenziazione megacariocita e isolamento delle cellule è descritto qui.

La differenziazione dei megacariociti e formazione delle piastrine da umani cavo emoderivati CD34 cellule+

Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells ...

megakaryocyte-differentiation-platelet-formation-from-human-cord

Research

JoVE Journal

Immunology and Infection

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells

19.5K Views.  University of New South Wales. Una popolazione altamente pura dei megacariociti sono ottenibili dal midollo derivate dal sangue CD34+ cellule. Un metodo per CD34+ differenziazione megacariocita e isolamento delle cellule è descritto qui.

Video: Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

Generazione di cellule T regolatrici indotte da cellule primarie umane T naive e memoria

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Ricerca

Immunologia e infezioni

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

Applicazione clinica della<em&gt; Bella Addormentata</em&gt; Artificiale e cellule presentanti l&#39;antigene per modificare geneticamente le cellule T da sangue del cordone ombelicale e periferico

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

In tempo reale di immagine degli eterotipica piastrine-neutrofili interazioni sull&#39;endotelio Attivato durante l&#39;infiammazione vascolare e la formazione di trombi in topi vivi

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

Isolamento di cellule dendritiche mieloidi e cellule epiteliali di timo umano

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

Generazione di Human alloantigene-specifiche cellule T da sangue periferico

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are used for host defense. For the quantitative determination of the functional status of the humoral immune response of an individual, both serum Abs and circulating ASCs are commonly measured as functional readouts. In humans, peripheral blood is the most convenient and readily accessible sample that can be used for the determination of the humoral immune response elicited by host B cells. Distinct B-cell subsets, including ASCs, can be isolated directly from peripheral blood via selection with lineage-specific Ab-conjugated microbeads or via cell sorting with flow cytometry. Moreover, purified na&#239;ve and memory B cells can be activated and differentiated into ASCs in culture. The functional activities of ASCs to contribute to Ab secretion can be quantified by ELISpot, which is an assay that converges enzyme-linked immunoabsorbance assay (ELISA) and western blotting technologies to enable the enumeration of individual ASCs at the single-cell level. In practice, the ELISpot assay has been increasingly used to evaluate vaccine efficacy because of the ease of handling of a large number of blood samples. The methods of isolating human B cells from peripheral blood, the differentiation of B cells into ASCs in vitro, and the employment of ELISpot for the quantification of total IgM- and IgG-ASCs will be described here.

Human peripheral blood is commonly used for the assessment of the humoral immune response. Here, the methods for isolating human B cells from peripheral blood, differentiating human B cells into antibody (Ab)-secreting B cells (ASCs) in culture, and enumerating the total IgM- and IgG-ASCs via an ELISpot assay are described.

L&#39;isolamento, la differenziazione e quantificazione di Human anticorpo-secernenti cellule B da sanguigno: ELISpot come una lettura funzionale del umorale immunità

The hallmark of humoral immunity is to generate functional ASCs, which synthesize and secrete Abs specific to an antigen (Ag), such as a pathogen, and are ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Generazione di derivate da monociti cellule dendritiche umane da sangue intero

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

La differenziazione in vitro di cellule CD4 + umana FOXP3 + indotta da T regolatorie (iTregs) da cellule T CD4 Naïve utilizzando un protocollo TGF-β contenenti

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

Sviluppo e validazione di una singola molecola ultrasensibile di matrice digitale analisi enzima-collegata dell'immunosorbente per umana dell'interferone-α

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

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 ...