La ricerca nei laboratori è finanziato dalla Fondazione Fritz-Bender, Monaco di Baviera, Germania (a TD) e l&#39;Università di Witten / Herdecke assegno di ricerca interno (a W.-KL). Vorremmo ringraziare il Prof. Dr. Dr. Frank Thévenod (Istituto di Fisiologia, Fisiopatologia e Tossicologia, Università di Witten / Herdecke) per gli utili suggerimenti.

1. Preparazione dei reagenti e soluzioni <ol><li> Preparare la soluzione di un sale di Hank modificato bilanciata (HBSS) (in mM: 136,9 NaCl, KCl 5,37, 0,34 Na 2 HPO 4, 0.44 KH 2 PO 4, 4.17 NaHCO3, pH 7,2, senza rosso fenolo). Conservare a 4 ° C. </li><li> Fai 1,5 M CaCl 2 e 2 M 4- (2-idrossietil) -1 acido-piperazineethanesulfonic (HEPES) (pH 7.2) soluzione madre con acqua distillata. </li><li> Preparare una soluzione 250 mM magazzino di probenecid. Per la forma di acido libero (spesso chiamato insolubile in acqua), sciogliere in polvere probenecid in NaOH 1N, riempire a tre quarti del volume finale con acqua distillata e titolare lentamente a pH 7,4 con HC1. Conservare a 4 ° C. </li><li> Sciogliere membrana permeabile colorante fluorescente calcio vincolante anidra dimetilsolfossido (DMSO) ad una concentrazione magazzino di 1 mm. Conservare a -20 ° C al buio. </li><li> Prima di ogni esperimento, appena preparare-probenecid contenente HBSS flusso (PHF) buffer combinando modificato con HBSS (concentrazioni finali) 1.5 mM CaCl 2, HEPES 10 mM, pH 7,2, siero fetale bovino al 5%, 2 mM probenecid, e 5,55 mM glucosio. Riscaldare a 37 ° C. </li></ol> Cultura 2. Cella <ol><li> Tavola 2.5 x 10 5 cellule renali prossimali tubulari (WKPT-0293 Cl.2) per pozzetto di un piatto 6 pozzetti o 35 millimetri nel terreno di coltura standard e di crescere per 2 giorni che raggiungono una confluenza di circa il 70%. </li></ol> 3. Calcio Dye Loading <ol><li> Per ogni piatto bene o, mescolare 1 ml di terreno di coltura contenente siero fetale bovino 5%, 4 celle mM permeabile calcio colorante vincolante e probenecid mM 2. </li><li> Sostituire il terreno di coltura con 1 ml di miscela di colorante di caricamento in ciascun pozzetto ed incubare a 37 ° C per 30 minuti in un incubatore umidificato con 5% di CO 2. Coprire la piastra con un foglio di alluminio per evitare lo sbiancamento del indicatore fluorescente. </li></ol> 4. Preparazione di CeLLS per Citometria a Flusso <ol><li> Soluzione 0,05% di acido tripsina-etilendiamminotetraacetico Warm (EDTA) a 37 ° C. </li><li> Aspirare calcio soluzione colorante di caricamento da ogni pozzetto e aggiungere 1 ml di tripsina, lungo la parete del pozzo. </li><li> Incubare a 37 ° C fino a quando tutte le celle sono staccati. </li><li> Trasferire le cellule in una provetta da 1,5 ml e pellet per centrifugazione in una microcentrifuga a 10.000 g per 1 min. </li><li> Aspirare accuratamente il sopranatante senza disturbare il pellet. </li><li> Lavare le cellule con l&#39;aggiunta di 1 ml di tampone PHF e risospendere pipettando su e giù. </li><li> Agglomerare cellule per centrifugazione a 10.000 g per 1 min. </li><li> Ripetere i punti da 4.6. e 4.7. altre due volte. </li><li> Risospendere le cellule in un volume finale di 500 microlitri di buffer PHF e utilizzare le cellule per la sperimentazione entro 1 ora. </li><li> Opzionali: le cellule di conteggio per determinare la concentrazione cellulare e utilizzare 5 x 4-01 ottobre x 10 5 cellule per la misurazione. </li></ol> 5. Flusso CytImpostazione ometer <ol><li> Avviare il citometro a flusso, aprire il cassetto fluidica e capovolgere l&#39;interruttore valvola di sfiato per aumentare la pressione d&#39;aria del serbatoio del liquido guaina. </li><li> &quot;Prime&quot; lo strumento per rimuovere eventuali bolle d&#39;aria che risiede all&#39;interno delle camere d&#39;aria e la camera di flusso. </li><li> Nel software di citometria a flusso, aperte due finestre dot plot. <ol><li> Il primo diagramma a punti richiede luce diffusa in avanti (FSC) per il parametro X e laterale della luce diffusa (SSC) per il parametro Y per controllare la qualità delle cellule all&#39;interno del campione attraverso la loro dimensione e la loro granularità, rispettivamente. </li><li> Il secondo diagramma a punti misura l&#39;intensità di fluorescenza del colorante di calcio-bound. Selezionare il canale di rilevamento di fluorescenza appropriato per il parametro Y usando una scala logaritmica e il tempo in secondi per il parametro X su una scala lineare. </li></ol></li><li> Impostare la portata di &#39;alto&#39;. </li></ol> 6. Citometria a flusso Measurement <ol><li> Eseguire le misurazioni a temperatura ambiente. In un citometria a flusso provetta (11,5 x 75 mm), aggiungere 480 microlitri di buffer PHF. </li><li> Aggiungi sospensione cellulare 20 ml e brevemente vortice per mescolare. </li><li> Spostare il braccio di supporto del tubo di lato e posizionare il tubo campione sul tubo di iniezione del campione fino a quando si forma una tenuta ermetica. Riportare il braccio di supporto del tubo nella posizione originale centrata. </li><li> Ottimizzare misurazione regolando il canale di fluorescenza tale che l&#39;intensità di fluorescenza media del campione è di circa 10 2 sul asse y. Salvare e utilizzare per gli esperimenti successivi. </li><li> Per avviare un esperimento, ripetere i passaggi 6,1-6,3. </li><li> Record di fluorescenza di base per 50 sec. </li><li> Pausa misurazione, rimuovere la provetta, aggiungere composti di interesse, vortex brevemente e ritornare al tubo di iniezione del campione. Questo passaggio non deve durare più di 15 secondi. </li><li> Riprendi misurazione e continuare per un totale di 204,8 sec. </li><li> Utilizzare un i di calcioonophore, come ionomicina (10 mM), e un chelante del calcio, come il glicole etilenico tetraacetico (EGTA), MnCl 2 o CoCl 2 (2 mM), per i controlli, rispettivamente positivo e negativo. </li></ol> Analisi 7. I dati <ol><li> Analizzare i dati utilizzando un software dedicato citometria a flusso per l&#39;analisi offline, come WinMDI (Windows Multiple Document Interface per citometria a flusso), un pacchetto software gratuito sviluppato da Joe Trotter presso l&#39;Istituto Scripps, Citometria a flusso Nucleo strumento. </li><li> Aprire un diagramma a punti con i parametri SSC e FSC. </li><li> Selezionare la regione di celle per l&#39;analisi con lo strumento &quot;Regioni&quot; (tasto destro del mouse sopra l&#39;immagine) di escludere le cellule morte o detriti cellulari si trovano in basso a sinistra dall&#39;analisi dei dati (Figura 1). </li><li> Analisi Quadrant (Figura 2) <ol><li> Aprire una trama densità con tempo in ascisse e l&#39;intensità di fluorescenza in asse y. Utilizzare tutti gli eventi. </li><li> Noiuna e la stessa regione di gating come in 7.3. </li><li> Quantificare i dati utilizzando funzione &quot;Quadrant&quot;. </li><li> Regolare i separatori quadrante in modo che la linea verticale viene posizionato al momento della aggiunta del composto e la linea orizzontale è posta al centro della fluorescenza basale nei controlli. Assicurarsi che il posizionamento dei separatori quadranti è uguale in tutti i campioni. </li><li> La percentuale di cellule in ciascun quadrante viene visualizzato in ciascun angolo (Figura 2). Confrontare il quadrante in alto a destra (UR) tra i campioni. </li></ol></li><li> Analisi Istogramma (Figura 3) <ol><li> Aprire i dati di controllo in una finestra istogramma con intensità di fluorescenza sul asse x ed eventi sul asse y. Utilizzare tutti gli eventi. </li><li> Per una visione pittorica, aggiungere i dati di test da confrontare con la trama controllo come trame di sovrapposizione (andare in File → Apri File → selezionare File → Overlay). </li><li> Per l&#39;analisi quantitativa, unico istogramma vinceredows devono essere utilizzati. Porta la popolazione di cellule con la regione R1 dal punto 7.3. </li><li> Nel controllo non trattato, utilizzare la funzione marker (&quot;marker&quot;) per contrassegnare l&#39;area di analisi partendo dal punto medio tra il minimo e il massimo di fluorescenza del picco di controllo e termina alla massima intensità di fluorescenza. La percentuale di cellule in questo settore è calcolato. </li><li> Recuperare i dati dalla finestra delle statistiche. Ripetere l&#39;operazione per i rimanenti file di dati utilizzando la stessa area contrassegnata per l&#39;analisi. </li></ol></li><li> Analisi intensità di fluorescenza (Figura 4) <ol><li> Aprire un diagramma a punti nel tempo su l&#39;asse x e l&#39;intensità di fluorescenza in asse y. Utilizzare tutti gli eventi. </li><li> Attiva &quot;Modalità Kinetics&quot; e &quot;Overlay Kinetics Line&quot;. Una linea blu apparirà nella trama punto che rappresenta l&#39;intensità di fluorescenza media. </li><li> Quantificare l&#39;afflusso di calcio relativa mediante la creazione di più aree rettangolari. Ogni regione dovrebbe avere awLarghezza della merito &quot;50&quot;, che equivale a &quot;10 sec&quot;. Definire fino a 15 zone rettangolari. </li><li> Recupera quantificazione dalla finestra delle statistiche e copia in un foglio di calcolo. </li><li> Utilizzare i dati y-media per l&#39;analisi. Calcolare la media dei R2 a R6, che è equivalente a 20 a 50 sec. Questo è il valore di base. </li><li> Scegliere un intervallo di tempo adeguato per l&#39;analisi del flusso di calcio (dipende inizio, durata e intensità). </li><li> Calcolare i segnali di calcio di ogni regione all&#39;interno di questo intervallo di tempo rispetto al valore basale, cioè il segnale di fluorescenza prima aggiunta del composto, che è impostato al 100%. Determinare la media e la deviazione standard delle regioni inviare aggiunta del composto; questo è il segnale di calcio medio evocata dal composto. </li></ol></li></ol>

<ol>
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The authors have nothing to disclose.

Il calcio è un evento chiave in molteplici processi cellulari che agiscono come una seconda molecola di segnalazione messaggero e anche come mezzo per comunicare tra ER e mitocondri. La possibilità di misurare le variazioni delle concentrazioni di calcio citosolico libero è una tecnica utile che può applicarsi a tutti i settori della biologia cellulare. Ci sono vari metodi per rilevare segnali di calcio citosolico. Classicamente, i segnali provenienti da un indicatore di calcio raziometrica, come la Fura-2, sono mon...

Il calcio è un importante secondo messaggero responsabile della trasmissione dei fisiologica cellulare e segnali fisiopatologici 1. Le concentrazioni di calcio citosolico sono tenuti bassi attraverso l&#39;attività di pompe e scambiatori di calcio di calcio. Il / ATPasi del reticolo endoplasmatico calcio sarcoplasmatico (SERCA) riempie il reticolo endoplasmatico (ER) negozio di calcio come parte di un sistema di &quot;perdita della pompa&quot;, mentre l&#39;ATPasi della membrana plasmatica di calcio (PMCA) estrude calcio al compartimento extracellulare, sia in maniere ATP-dipendenti 2. Messaggeri fisiologici, come gli ormoni o neurotrasmettitori, trasmettono i loro segnali attraverso G-recettori accoppiati alle proteine, l&#39;attivazione della fosfolipasi C, che idrolizza fosfatidilinositolo 4,5-bisfosfato (PIP2) nella membrana plasmatica per generare diacilglicerolo e inositolo trifosfato (IP3) 3. Mentre diacilglicerolo rimane nella membrana plasmatica, IP3 diffonde nel citosol e si lega al recettore IP3s (IP3Rs), che sono ligandi attivano i canali del calcio, che si trova nella membrana ER e il calcio viene rilasciato dal negozio ER luminale si conclude con un aumento delle concentrazioni di calcio citosolico 4. Una via alternativa a calcio per raggiungere il citosol è attraverso i canali del calcio e scambiatori presenti nella membrana plasmatica. Crosstalk tra questi due comparti sono stati descritti: rilascio di calcio di calcio indotto (CICR) in cui il calcio extracellulare induce il rilascio di calcio dai depositi ER 5, e conservare Entry operato di calcio (Soče), dove lo svuotamento dei depositi ER viene rilevata da STIM e provoca l&#39;apertura di Orai canali del calcio nella membrana plasmatica per promuovere riempimento dei negozi ER 6. In condizioni fisiopatologiche, la risposta cellulare di calcio è un aumento di calcio citosolico liberalizzati e in assenza di stimolatori fisiologici 1. La risposta di calcio può essere effettuata in vari modi: prolungata calciodel segnale, ritardato la rimozione del calcio dal citosol, esaurimento delle riserve di calcio o modifiche localizzate del calcio. Inoltre, i mitocondri assumono un ruolo centrale nel buffer e liberando calcio 7. Prolungato e / o sovramassimale aumento di calcio citosolico porta alla morte cellulare. In realtà, il calcio è il più delle volte coinvolto nella risposta cellulare e fisiopatologico è un evento chiave in una vasta gamma di malattie, come le malattie neurodegenerative, le malattie cardiache, il cancro, le malattie delle ossa e malattie renali, che sono principalmente associato con la morte delle cellule e la perdita o l&#39;alterazione di organo o tessuto funzione 8-10. Inoltre, perturbazione dei segnali di calcio è stato collegato a adattamento cellulare e cambiamenti nelle funzioni e risposte cellulari. Tradizionalmente, segnali di calcio sono misurati con un indicatore di calcio fluorescenti carica negativa che viene caricato in cellule in una cella acetossimetil permeabili (AM) forma estere. Una volta spaccati da Esteras cellularies, gli indicatori fluorescenti rimangono all&#39;interno del compartimento intracellulare e aumentano di intensità di fluorescenza quando il calcio è legato. L&#39;indicatore di calcio più conosciuto e utilizzato è il raziometrico Fura-2 sviluppato da Roger Tsien e colleghi 11. Indicatori di calcio con differenti affinità per il calcio consentono diverse piscine di calcio da monitorare. Metodi di rilevamento includono l&#39;imaging cellulare dal vivo, lettore di micropiastre a fluorescenza e citometria a flusso. La cinetica relativamente veloce dei segnali di calcio (di solito nel giro di pochi secondi a minuti) rende l&#39;imaging cellulare dal vivo il metodo migliore per ottenere la maggior parte delle informazioni sulle caratteristiche del segnale calcio. A parte la cinetica rapida dei segnali di calcio (nel giro di millisecondi), l&#39;imaging cellulare dal vivo è un metodo migliore per studiare compartimentalizzazione cellulare di segnalazione di calcio all&#39;interno di una singola cella 12. Le concentrazioni di calcio assoluti possono essere calcolate determinando la fluorescenza minima e massima della ind di calcioicator aggiungendo un chelante del calcio e permeabilizzante cellule, rispettivamente, come descritto da Grynkiewicz et al. 11. L&#39;uso di citometria a flusso per misurare segnali di calcio è stato sviluppato nel 1980 in cellule immunologiche dove la possibilità di misurare più parametri, come l&#39;integrità della membrana e la separazione della popolazione cellulare, e il requisito di cellule in sospensione in combinazione con lo sviluppo di singola lunghezza d&#39;onda indicatori di calcio fatto citometria a flusso un metodo ideale e convenientdetection 13-15. In cellule aderenti, cellule vive fornisce più informazioni sulla segnalazione di calcio, soprattutto la cinetica, ma richiede una configurazione complicata comprendente un microscopio a fluorescenza, un sistema di perfusione, manutenzione dell&#39;ambiente cellulare, come la temperatura, e il software microscopio specializzato per acquisire e analisi dei dati. Metodi alternativi come ad esempio un lettore di micropiastre a fluorescenza o Pharmacolmezzi ogical attraverso l&#39;uso di chelanti del calcio sono incomparabili in termini di informazioni acquisite. Citometria a flusso sta diventando più ampiamente usato per monitorare calcio citosolico in cellule aderenti però, nella maggior parte degli studi, solo punto finale vengono effettuate misurazioni. Utilizzando il metodo sviluppato inizialmente da Gergely et al. In cellule B immunologiche 16, cambiamenti nella concentrazione di calcio libero citosolico di citometria a flusso con una cinetica in tempo reale e monitorare l&#39;intensità di fluorescenza di singole cellule provenienti da un campione di popolazione sono stati misurati con successo in cellule epiteliali tumorali e cellule staminali tumorali ibridi 17. Questo metodo è stato ulteriormente adattato per l&#39;uso in cellule epiteliali aderenti, che sono difficili da caricare con indicatori di calcio 18. Qui, utilizzando una linea cellulare epiteliale aderente immortalato derivato dal segmento S1 del tubulo prossimale rene di ratto (WKPT-0293 Cl.2) 19, descriviamo un ottimizzato m semplificatoetodo per la misurazione delle variazioni di concentrazione di calcio citosolico di citometria a flusso. Dal momento che molte cellule epiteliali in possesso di un certo numero di trasportatori organici per i composti anionici, carico di cellule con un indicatore di calcio può rivelarsi una sfida. Per impedire l&#39;efflusso di indicatori di calcio, probenecid, l&#39;inibitore prototipico dei trasportatori di anioni organici e originariamente sviluppato per diminuire l&#39;escrezione renale di penicillina 20, viene applicata simultaneamente nel mezzo di incubazione. Le cellule sono mantenute in monostrati di indicatore di calcio di carico, portati in segnali di sospensione e di calcio può essere rilevata immediatamente dopo l&#39;aggiunta composto.

<table border="0" cellpadding="0" cellspacing="0" width="735">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:79px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:321px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluo-3, AM</td>
			<td style="width:79px;">Invitrogen</td>
			<td style="width:119px;">F-1241&#160;</td>
			<td>Cell permeable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Probenecid</td>
			<td style="width:79px;">Sigma-Aldrich</td>
			<td style="width:119px;">P-8761</td>
			<td>Water insoluble, dissolve in 1 N NaOH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.05% Trypsin-EDTA (1X)</td>
			<td style="width:79px;">Invitrogen</td>
			<td style="width:119px;">25300-062</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ionomycin</td>
			<td style="width:79px;">Sigma-Aldrich</td>
			<td style="width:119px;">I9657</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Thapsigargin</td>
			<td style="width:79px;">Tocris Bioscience</td>
			<td style="width:119px;">1138</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tunicamycin</td>
			<td style="width:79px;">Sigma-Aldrich</td>
			<td style="width:119px;">T7765</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">FACSCalibur Flow Cytometer + CELLQuest software</td>
			<td style="width:79px;">Becton Dickinson</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Windows Multiple Document Interface for Flow Cytometry (WinMDI)</td>
			<td style="width:79px;">Joe Trotter, The Scripps Institute</td>
			<td style="width:119px;"></td>
			<td style="width:321px;">Please note that this is an older 16-bit application that reads FCS 2.0 compliant files but does not recognize FCS 3.0 digital data.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">WKPT-0293 Cl.2 rat kidney proximal tubule cell line</td>
			<td style="width:79px;"></td>
			<td style="width:119px;"></td>
			<td style="width:321px;">Made available by Dr. Ulrich Hopfer (Department of Physiology &#38; Biophysics, Case 
			Western Reserve University, Cleveland, OH)</td>
		</tr>
	</tbody>
</table>

cytosolic calcium measurements in renal epithelial cells by flow cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Per aumentare il calcio citosolico, due composti farmacologici sono stati applicati alle cellule renali prossimali tubulari (WKPT-0293 Cl.2) Pellicola colorante legame delle cellule di calcio permeabile, Fluo-3-AM. Campioni di controllo non trattate sono state caricate con calcio legame con colorante in presenza di probenecid e sono stati mescolati, ma senza l&#39;aggiunta di composti. Tapsigargina (TG) è un inibitore classica della pompa SERCA che porta ad una perdita di pronto soccorso e un conseguente aumento di cal...

Watch this Scientific Journal Video about Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry at JoVE.com

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

Il calcio è coinvolto in numerose vie di segnalazione fisiologici e fisiopatologici. Imaging cellulare dal vivo richiede attrezzature specializzate e può richiedere molto tempo. Un rapido, metodo semplice usando un citometro di flusso per determinare variazioni relative del calcio citosolico nelle cellule epiteliali aderenti portati in sospensione è stata ottimizzata.

Misure calcio citosolico nelle cellule epiteliali renali mediante citometria di flusso

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

cytosolic-calcium-measurements-renal-epithelial-cells-flow

Research

JoVE Journal

Biology

15.2K Views.  University of Witten/Herdecke.  Il calcio è coinvolto in numerose vie di segnalazione fisiologici e fisiopatologici. Imaging cellulare dal vivo richiede attrezzature specializzate e può richiedere molto tempo. Un rapido, metodo semplice usando un citometro di flusso per determinare variazioni relative del calcio citosolico nelle cellule epiteliali aderenti portati in sospensione è stata ottimizzata. 

Video: Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.

The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.

IP-FCM: Immunoprecipitazione rilevato mediante citometria di flusso

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

Ricerca

Biologia

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

Purificazione Citometria a flusso di cellule di topo Meiotic

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Isolamento di cromatina Purificazione RNA (disturbo)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Ottimizzato Metodi di modellazione e colorazione proliferazione per il monitoraggio divisione cellulare utilizzando coloranti Tracking Cellulari

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

Alta efficienza differenziazione delle cellule staminali pluripotenti umane di cardiomiociti e Caratterizzazione mediante citometria di flusso

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Analisi delle sospensioni cellulari isolate da tessuti solidi da Spectral citometria a flusso

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated with specialization, development and increase in cellular size. In plants, endoreduplication seems to facilitate the growth and expansion of certain tissues and organs. Among them is the tuber of potato (Solanum tuberosum), which undergoes considerable cellular expansion in fulfilling its function of carbohydrate storage. Thus, endoreduplication may play an important role in how tubers are able to accommodate this abundance of carbon. However, the cellular debris resulting from crude nuclear isolation methods of tubers, methods that can be used effectively with leaves, precludes the estimation of the tuber endoreduplication index (EI). This article presents a technique for assessing tuber endoreduplication through the isolation of protoplasts while demonstrating representative results obtained from different genotypes and compartmentalized tuber tissues. The major limitations of the protocol are the time and reagent costs required for sample preparation as well as relatively short lifespan of samples after lysis of protoplasts. While the protocol is sensitive to technical variation, it represents an improvement over traditional methods of nuclear isolation from these large specialized cells. Possibilities for improvements to the protocol such as recycling enzyme, the use of fixatives, and other alterations are proposed.

The protocol described herein is a method for measuring endoreduplication within tubers of potato (Solanum tuberosum). It includes plasmolysis and protoplast extraction steps to decrease the noise and debris in downstream flow cytometric analysis.

Endoreduplicazione tramite flusso Cytometry di tubero isolato protoplasti di misura

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, over 90% of which is due to macropinocytosis. In addition, many of the known core components of mammalian macropinocytosis are also present, making it an excellent model system for studying macropinocytosis. Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment.
Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. The method can also be adapted to measure membrane uptake and phagocytosis of fluorescent beads or bacteria.
This method was designed to allow measurement of fluid uptake by Dictyostelium in a high-throughput, labor and resource efficient manner. It allows simultaneous comparison of multiple strains (e.g. knockout mutants of a gene) and conditions (e.g. cells in different media or treated with different concentrations of inhibitor) in parallel and simplifies time-courses.

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Macropinocytosis, large-scale non-specific fluid uptake, is important in many areas of clinical biology including immunology, infection, cancer, and neurodegenerative diseases. Here, existing techniques have been adapted to allow high-throughput, single-cell resolution measurement of macropinocytosis in the macropinocytosis model organism Dictyostelium discoideum using flow cytometry.

Misurazione di alto-rendimento di Dictyostelium discoideum Macropinocitosi tramite flusso Cytometry

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.

Rilevamento e caratterizzazione dell'autoassemblaggio proteico in vivo da parte della Cytometria di flusso

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from ...

Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production of all mature blood cells. In recent years cellular metabolism has emerged as a crucial regulator of HSC function and fate. We have previously demonstrated that modulation of mitochondrial metabolism influences HSC fate. Specifically, by chemically uncoupling the electron transport chain we were able to maintain HSC function in culture conditions that normally induce rapid differentiation. However, limiting HSC numbers often precludes the use of standard assays to measure HSC metabolism and therefore predict their function. Here, we report a simple flow cytometry assay that allows reliable measurement of mitochondrial membrane potential and mitochondrial mass in scarce cells such as HSCs. We discuss the isolation of HSCs from mouse bone marrow and measurement of mitochondrial mass and membrane potential post ex vivo culture. As an example, we show the modulation of these parameters in HSCs via treatment with a metabolic modulator. Moreover, we extend the application of this methodology on human peripheral blood-derived T cells and human tumor infiltrating lymphocytes (TILs), showing dramatic differences in their mitochondrial profiles, possibly reflecting different T cell functionality. We believe this assay can be employed in screenings to identify modulators of mitochondrial metabolism in various cell types in different contexts.

Here we describe a reliable method to measure mitochondrial mass and membrane potential in ex vivo cultured hematopoietic stem cells and T cells.

Misurazione della massa mitocondriale e del potenziale della membrana nelle cellule staminali ematopoietiche e t-cellule da parte della citometria di flusso

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production ...