Ringraziamo Laurie Destroismaisons e Sarah Peyrard per un supporto tecnico e Dr.Alexandre Prat per l&#39;accesso al citofluorimetro. Vorremmo inoltre ringraziare il dottor Timothy Miller per il suo contributo per quanto riguarda la rimozione della mielina dai preparativi. Questo lavoro è stato supportato dal Canadian Institutes of Health Research (CIHR) Neuromuscular Research Partnership, Canadian Foundation for Innovation, ALS Society of Canada, la Fondazione Frick per la SLA Research, CHUM Fondazione e Fonds de la Recherche en Santé du Québec (CVV). Sia CVV e NA sono studiosi di ricerca del Fonds de la Recherche en Santé du Québec e CIHR nuovi ricercatori. SP è sostenuto dalla Tim Noël Studentship dalla ALS Society of Canada.

Animali utilizzati in questo studio sono stati trattati in stretta conformità ad un protocollo (N08001CVsr) approvato dal du Centre Hospitalier de l&#39;Université de Montréal (CRCHUM) Comitato istituzionale Centre de Recherche per la protezione degli animali che segue gli standard nazionali, come delineato dalla Canadian Consiglio su Animal Care (CCAC). Preparare tutti i reagenti necessari per eseguire questo protocollo (Tabella 1). Tutti gli altri dettagli riguardanti attrezzature, forniture e fornitori possono essere trovati nella lista dei materiali. <img alt="Tabella 1" fo:content-width="5in" src="/files/ftp_upload/51887/51887table1.jpg" width="500" /> Tabella 1 Buffer Composizioni. 1 Raccolta di Rat Spinal Cord <ol><li> Profondamente anestetizzare il ratto (Sprague Dawley) con il 4% isoflorane. Verificare l&#39;anestesia da una mancanza di riflesso su di schiacciamento di the zampa anteriore. Euthanize il ratto per decapitazione mediante ghigliottina. Questo metodo di eutanasia è preferito rispetto ad altri, che potrebbero distorcere il midollo spinale. </li><li> Tagliare la pelle della schiena per esporre la colonna vertebrale. Tagliare la colonna vertebrale con le forbici osso appena sopra l&#39;osso pelvico. Visualizzare l&#39;apertura della colonna vertebrale. </li><li> Inserire una siringa da 10 ml, con una pipetta punta di 200 microlitri (collegato tramite la fusione poco più di fiamma), riempita con tampone fosfato (PBS), nella colonna vertebrale. </li><li> Lavare il midollo spinale, applicando una quantità media di pressione allo stantuffo. </li><li> Se è presente del sangue sul midollo spinale, risciacquare con PBS prima di procedere alla fase successiva. NOTA: Questo metodo è stato validato anche per il cervello e il fegato. Se tra cui questi tessuti, raccogliere la metà del cervello dal ratto eutanasia e un pezzo di fegato pari in peso al midollo spinale. Tutti gli altri passaggi rimangono identici. </li></ol> 2 Isolamento del midollo spinale Mitochondria (Adattato da Vande Velde et al. 4) <ol><li> Raccogliere tutto il midollo spinale intatta e posto in 5 ml omogeneizzatore vetro con 5 volumi (~ 3,25 ml) Omogeneizzazione Buffer (HB). Per il recupero ottimale dei mitocondri isolati, eseguire tutti i passaggi su ghiaccio o in camera fredda. Omogeneizzare i tessuti a mano fino a quando rimangono senza grandi pezzi di tessuto, circa otto colpi. Mettere omogenato in due (2 ml) o tre (1,7 ml) microprovette. Centrifuga 1.300 xg per 10 min a 4 ° C in una microcentrifuga da banco. </li><li> Recuperare il surnatante e posto in un tubo ultracentrifuga 5 ml. Aggiungere 750 ml (~ 0,5 volumi) HB alla provetta-pellet contenente e risospendere delicatamente il pellet. Ripetere la centrifugazione e risospensione passi altre due volte. Pool tutti i surnatanti (S1a e S1b) nella stessa 5 ml tubo ultracentrifuge sopra. Questo passaggio serve per eliminare piccoli detriti. </li><li> Centrifugare riunito S1 utilizzando un&#39;ultracentrifuga dotato di marciume oscillante secchioo e centrifugare a 17.000 xg per 15 min a 4 ° C. </li><li> Tenere surnatante (S2) per l&#39;ulteriore elaborazione se la frazione citosolica è di interesse (ad esempio per le analisi Western blot). Risospendere il pellet (P2), la frazione mitocondriale grezzo, in 4 ml HB + KCl 50 mM. Centrifugare 17.000 xg per 15 min a 4 ° C in un rotore oscillante. Eliminare il supernatante e risospendere delicatamente il pellet (P3) in 800 ml HB. NOTA: I mitocondri vengono lavati con HB + 50 mM KCl per rimuovere eventuali contaminanti mitocondrialmente associati non specifici. </li><li> In un nuovo 5 ml tubo Ultracentrifuge, aggiungere esattamente 800 ml di sedimento risospeso (P3). </li><li> Per aggiungere questo tubo, 200 ml di Iodixanolo (media gradiente di densità), creando così una concentrazione finale del 12% Iodixanolo. Miscelare il contenuto della provetta delicatamente, ma accuratamente mediante pipettaggio con un P1000. Aggiunta di Iodixanolo prima, e poi pellet risospeso (P3) può essere preferibile per facilitare un&#39;accurata miscelazione. Centrifugare in un ultracentrifuge dotato di un rotore benna oscillante a 17.000 xg per 15 min a 4 ° C. </li><li> NOTA: Fegato non contiene mielina e, pertanto, questo passaggio non è necessario se solo il fegato è in fase di elaborazione. Tuttavia, se il fegato sta per essere eseguita in concomitanza con mitocondri del SNC, si raccomanda di trattare tutti i tessuti allo stesso modo. </li><li> Aspirare lo strato di mielina nella parte superiore del tubo e con attenzione rimuovere e scartare il surnatante. Pellet potrebbe essere allentato. Risospendere il pellet in 4 ml di HB. Centrifugare nuovamente a 17.000 xg per 10 min a 4 ° C. Eliminare il supernatante e risospendere il pellet in 4 ml di HB. Ripetere la centrifugazione e rimuovere il surnatante. </li><li> Risospendere il pellet finale (P7) in 100-200 ml HB e trasferire in una provetta da 1,7 ml microcentrifuga. Questo campione contiene mitocondri isolati. </li><li> Procedere alla proteina quantificazione. Diluire i campioni e la curva standard nel 2% sodio dodecil solfato (SDS) per assicurare un&#39;adeguata solubilizzazione dei mitocondri During proteina quantificazione. </li></ol> 3. immunomarcatura di mitocondri isolati per Citometria a flusso <ol><li> Per ogni mix colorazione da testare, pipetta 25 mg di mitocondri isolati in una provetta da 1,7 ml microcentrifuga. Includere un campione senza macchia in ogni esperimento; e per ogni anticorpo, essere sicuri di includere un campione per il controllo isotipico appropriato. </li><li> Centrifugare a 17.000 xg per 2 min a 4 ° C in una microcentrifuga da banco. </li><li> Rimuovere il surnatante e risospendere mitocondri isolati in 50 microlitri mitocondri Buffer (Buffer M) supplementato con 10% di BSA libera acidi grassi per 15 min a 4 ° C (blocco passo). NOTA: Durante l&#39;etichettatura, effettuare incubazioni in frigorifero a 4 ° C. </li><li> Aggiungere l&#39;anticorpo primario (coniglio anti-MFN2, 20 mg per ml) per tubo e incubare per 30 minuti a 4 ° C. NOTA: Determinare la concentrazione ottimale di ciascun anticorpo empiricamente mediante titolazione. A causa della variabilità della concentrazione e / o purezza, diversi lotti dello stesso anticorpo dello stesso produttore possono portare a risultati diversi; pertanto è necessaria titolazione per ciascun nuovo lotto di anticorpi. </li><li> Lavare anticorpo primario non legato: Centrifugare a 17.000 xg per 2 min a 4 ° C. Rimuovere il surnatante e risospendere delicatamente il pellet in 200 ml M Buffer. Centrifugare a 17.000 xg per 2 min a 4 ° C. Rimuovere il surnatante e risospendere il pellet in 50 microlitri M tampone. </li><li> Aggiungere l&#39;anticorpo secondario (Donkey anti-coniglio IgG ficoeritrina (PE), 0,5 mg per ml) di tubo e incubare i campioni per 30 minuti a 4 ° C, al riparo dalla luce. </li><li> Lavare anticorpo secondario non legato: Centrifugare a 17.000 xg per 2 min a 4 ° C. Rimuovere il surnatante e risospendere il pellet in 200 ml M Buffer. Centrifugare a 17.000 xg per 2 min a 4 ° C. Rimuovere il surnatante e risospendere il pellet in 500 microlitri M tampone. </li><li> Per assicurare gli eventi sono in realtà mitocondri, macchia isolato mitocondri concolorante mitocondri specifico fluorescente per 15 minuti a temperatura ambiente, al riparo dalla luce. Se si desidera la colorazione di altri parametri funzionali (potenziale transmembrana mitocondriale o di produzione superossido), passare al punto 4 In caso contrario, passare al punto 5 per l&#39;acquisizione. NOTA: È importante verificare che lo spettro di emissione della anticorpo secondario è compatibile con quella dei coloranti funzionali. Ad esempio, se la verifica purezza mitocondriale con un colorante commerciale con proprietà spettrali simili a potenziale transmembrana con FITC e tetramethylrhodamine estere metilico (TMRM), un anticorpo secondario praticabile sarebbe allophycocyanin (APC: Ex 650 nm / em 660 nm). Aggiungere controlli compensazioni, cioè, un campione immunolabeled o tinto con un unico fluoroforo, quando applicabile. </li><li> Trasferire in un tubo adatto per il flusso di carico citometro. (Per facilitare la piccola dimensione del campione, un tubo di microtitolazione è collocato all&#39;interno 3 ml citofluorimetro tubo.) Conservare i campioni in ghiaccio e procedere immediatamente allacitofluorimetro per l&#39;acquisizione. </li></ol> 4 Saggio mitocondriale potenziale transmembrana mitocondriale superossido e Produzione per Citometria a flusso <ol><li> Verificare che i mitocondri isolati hanno un potenziale transmembrana intatto dalla colorazione con 100 nM TMRM (Ex 548 nm / 574 nm Em) 5, al punto 3.8, per 15 minuti a temperatura ambiente, al riparo dalla luce. Per il confronto del potenziale transmembrana tra i campioni e popolazioni, l&#39;uso di concentrazioni inferiori / non-tempra di TMRM (da 1 a 30 Nm), può essere più appropriato 6. <ol><li> Come controllo per TMRM colorazione, macchia mitocondri isolati con 100 nM TMRM in presenza di 100 mM carbonilico cianuro m-cloro fenil idrazina (CCCP), un disaccoppiatore mitocondriale che depolarizzare mitocondri. La concentrazione di CCCP necessaria per depolarizzare i mitocondri può essere inferiore se si utilizzano più basse concentrazioni di colorante. </li></ol></li><li> Verificare che i mitocondri isolati producono superossido mitocondriale da staining con un appropriato indicatore superossido mitocondriale 7, anche al punto 3.8, per 15 minuti a temperatura ambiente, al riparo dalla luce. <ol><li> Come controllo per la produzione di superossido mitocondriale, macchia mitocondri isolati con colorante in presenza di 10 mM Antimicina A, un inibitore del complesso III della catena respiratoria che aumenterà la produzione di superossido mitocondriale. </li></ol></li></ol> 5. acquisizione e analisi dei immunolabeled mitocondri isolati da Citometria a flusso <ol><li> Strumento costituito: tensioni Passare dalla lineare alla modalità log per facilitare l&#39;analisi dei mitocondri isolati e tensioni previste (FSC: 450; SSC: 250). Assicurarsi che gli eventi sono raccolti in FSC modo A (area) così come FSC-W (larghezza) e FSC-H (altezza), per poter escludere doppietti (cioè due eventi, passano attraverso il rivelatore allo stesso tempo ) nella raccolta di post-analisi dei dati. Impostare il numero di eventi da raccogliere a 100.000. Acquisire i controlli di compensazione, Se applicabile. </li><li> Acquisizione dati: Prima di acquisizione dei dati, evitare vortex campioni. Invece Mescolare agitando leggermente il tubo. Inizialmente la raccolta di eventi a bassa pressione, durante gating. Gate popolazione totale. Regolare le tensioni di istogrammi di conseguenza, di solito il picco del campione non colorato corrisponderà al secondo decennio (10 2). Una volta che cancelli sono stabiliti, ei campioni vengono elaborati, la pressione può essere commutata in alto. </li><li> Analisi: Visualizza doppietti tracciando FSC-W contro FSC. Identificare canottiere e doppiette. Gate canottiere. Selezionare la popolazione mitocondriale da gating su eventi che sono macchiati positivamente con un colorante mitocondriale selettivo. </li><li> Determinare l&#39;etichettatura sfondo da controllo isotipico. Utilizzando il campione di controllo isotipico, determinare la percentuale della popolazione di etichettatura mitocondriale positivo per gli anticorpi MFN2. </li></ol>

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E &#39;sempre più evidente che i mitocondri sono i principali attori sia normale fisiologia e patologia. Mentre immunoblotting può determinare quali proteine ​​si trovano all&#39;interno dei mitocondri né sulla superficie mitocondriale in una certa condizione, questo metodo relazioni sulla media dell&#39;intera popolazione. Questo metodo non può fornire informazioni sulle abbondanze relative delle sottopopolazioni mitocondriali o sottoinsiemi. Mentre è stato precedentemente ipotizzato che tutti i mitocondri son...

I mitocondri sono organelli altamente dinamiche che subiscono vari cicli di fissione e fusione, sono trasportati ai siti di domanda di alta energia e di rispondere rapidamente agli stimoli fisiologici 1. Poiché è sempre più riconosciuto che i mitocondri all&#39;interno di diversi tessuti, anche diversi compartimenti cellulari, hanno profili funzionali distinti, nuovi metodi sono necessari per identificare questi sottoinsiemi mitocondriali distinti. Microscopia fornisce un mezzo attraverso il quale i singoli mitocondri possono essere visualizzati e la presenza di una proteina in o nei mitocondri può essere determinato mediante immunofluorescenza 2. Tuttavia, l&#39;analisi quantitativa con questo metodo è laborioso ed è più adatto per gli esperimenti che utilizzano linee cellulari immortalizzate o primari. Lo studio dei mitocondri individuale derivate da tessuti è molto più difficile e la maggior parte dei metodi non consentono una facile identificazione di sottoinsiemi mitocondriali in concomitanza con la valuzione della funzione mitocondriale 3. Per far fronte a questo ostacolo, un nuovo metodo per immunolabel mitocondri isolati da tessuti di roditori e successivamente analizzati mediante citometria a flusso è stato sviluppato. Questo permette la rapida individuazione e la quantificazione delle proteine ​​localizzate alla membrana esterna mitocondriale, che rispetto all&#39;analisi al microscopio, è molto meno laborioso e permette l&#39;analisi di migliaia di mitocondri in un singolo campione. Questo test può essere applicato per controllare il destino e la quantità relativa di proteine ​​di membrana esterna mitocondriale che si pensa siano costitutivamente presente alla mitocondri, l&#39;assunzione di proteine ​​alla superficie mitocondriale, o la rilevazione di proteine ​​mislocalized ai mitocondri in condizioni patologiche. Inoltre, l&#39;incorporazione di coloranti convenzionali indicatori fluorescenti consente la valutazione simultanea di alcuni aspetti della funzione mitocondriale in mitocondriale distintosottopopolazioni.

<table border="0" cellpadding="0" cellspacing="0" width="1276">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Name of Material/ Equipment</td>
			<td style="width:349px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:543px;">Comments/Description</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:267px;">Rats</td>
			<td style="width:349px;">Charles River</td>
			<td style="width:119px;">Strain code 400</td>
			<td style="width:543px;">Adult (9 weeks to 18 weeks) male or female rats can be used for the isolation protocol. Weight of rats is dependent on gender and age (males between 300 to 500 g and females between 200 to 350 g) are typically used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Dounce homogenizer</td>
			<td style="width:349px;">Kontes Glass Co.&#160;</td>
			<td style="width:119px;">885450-0022</td>
			<td>Duall 22</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Microcentrifuge</td>
			<td style="width:349px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td>Sorvall Legend Micro 17 R</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Ulara-Clear Ultracentrifuge tubes</td>
			<td style="width:349px;">Beckman Coultier</td>
			<td style="width:119px;">344057</td>
			<td>Transparent, thin walled&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Sorvall Ultracentrifuge</td>
			<td style="width:349px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td>Sorvall WX UltraSeries</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">AH-650 rotor and buckets&#160;&#160;</td>
			<td style="width:349px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:267px;">Opti-prep</td>
			<td style="width:349px;">Axis-Shield</td>
			<td style="width:119px;">1114542</td>
			<td>Iodixanol, density gradient medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Fatty acid free Bovine Serum Albumin</td>
			<td style="width:349px;">Sigma</td>
			<td style="width:119px;">A8806</td>
			<td>Must be fatty acid free for mitochondria</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Sodium succinate dibasic hexahydrate&#160;&#160;</td>
			<td style="width:349px;">Sigma</td>
			<td style="width:119px;">S9637</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:267px;">Rabbit anti-Mitofusin2 antibody</td>
			<td style="width:349px;">Sigma</td>
			<td style="width:119px;">M6319</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:267px;">Rabbit IgG</td>
			<td style="width:349px;">Jackson Immuno Research&#160;</td>
			<td style="width:119px;">011-000-003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Anti-Rabbit IgG PE</td>
			<td style="width:349px;">eBioscience</td>
			<td style="width:119px;">12-4739-81</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Micro titer tube</td>
			<td style="width:349px;">Bio-Rad</td>
			<td style="width:119px;">223-9391</td>
			<td>For sample acquisition by flow cytometry&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">MitoTrackerGreen (MTG)</td>
			<td style="width:349px;">Invitrogen</td>
			<td style="width:119px;">M7514</td>
			<td>100 nM: Ex490 nm/Em516 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">TMRM</td>
			<td style="width:349px;">Invitrogen</td>
			<td style="width:119px;">T668</td>
			<td>100 nM: Ex548 nm/Em574 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">CCCP</td>
			<td style="width:349px;">Sigma</td>
			<td style="width:119px;">C2759</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">MitoSOX Red</td>
			<td style="width:349px;">Invitrogen</td>
			<td style="width:119px;">M36008</td>
			<td>5 &#181;M: Ex540 nm/Em600 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Antimycin A</td>
			<td style="width:349px;">Sigma</td>
			<td style="width:119px;">A8874</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">LSR II flow cytometer</td>
			<td style="width:349px;">BD</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">BS FACSDiva Software</td>
			<td style="width:349px;">BD</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">FlowJo</td>
			<td style="width:349px;">TreeStar Inc.&#160;</td>
			<td style="width:119px;"></td>
			<td>Software used for analysis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">BCA protein assay kit&#160;&#160;</td>
			<td style="width:349px;">Pierce/Thermo Scientific</td>
			<td style="width:119px;">23225</td>
			<td style="width:543px;">Bradford assay is not recomended as it is not compatible with high concentrations of SDS</td>
		</tr>
	</tbody>
</table>

immunodetection of outer membrane proteins by flow cytometry of isolated mitochondria

Metodi per rilevare e monitorare mitocondriali componenti proteiche della membrana esterna nei tessuti animali sono di vitale importanza per lo studio della fisiologia mitocondriale e fisiopatologia. Questo protocollo descrive una tecnica in cui i mitocondri isolati da tessuto roditori sono immunolabeled e analizzati mediante citometria a flusso. I mitocondri sono isolate dal midollo spinale di roditori e sottoposti ad una fase di arricchimento rapido in modo da rimuovere mielina, un importante contaminante di frazioni mitocondriali preparate da tessuto nervoso. Mitocondri isolati vengono poi etichettati con un anticorpo di scelta e di un anticorpo secondario coniugato fluorescente. L&#39;analisi per flusso citometria verifica la relativa purezza delle preparazioni mitocondriali da colorazione con un colorante specifico mitocondriale, seguita da rilevamento e la quantificazione di proteine ​​immunolabeled. Questa tecnica è rapido, quantificabile e ad alta velocità, consentendo l&#39;analisi di centinaia di migliaia di mitocondri per campione. È applicabile per valutare romanzo proteins sulla superficie mitocondriale in condizioni fisiologiche normali, così come le proteine ​​che possono diventare mislocalized a questo organello durante patologia. È importante sottolineare che questo metodo può essere accoppiato a coloranti indicatori fluorescenti a riferire su determinate attività di sottopopolazioni mitocondriali ed è fattibile per i mitocondri del sistema nervoso centrale (cervello e midollo spinale), così come il fegato.

I mitocondri derivate da midollo spinale di ratto possono essere immunolabeled con un anticorpo mirato al Mitofusin2 (MFN2), una proteina coinvolta nella fusione della membrana esterna dei mitocondri 8. Dopo aver isolato e marcatura con un anticorpo specifico MFN2 e un anticorpo secondario coniugato fluorescente, mitocondri vengono elaborati mediante citometria di flusso (Figura 1). Dopo l&#39;acquisizione dei dati, i campioni vengono analizzati mediante citometria a flusso software di analis...

Watch this Scientific Journal Video about Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria at JoVE.com

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Qui descritto è un metodo per rilevare e quantificare proteine ​​di membrana mitocondriale esterna da immunomarcatura di mitocondri isolati da tessuto roditore e l&#39;analisi mediante citometria a flusso. Questo metodo può essere esteso a valutare aspetti funzionali di sottopopolazioni mitocondriali.

Immunolocalizzazione della membrana esterna proteine ​​mediante citometria di flusso di mitocondri isolati

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

immunodetection-outer-membrane-proteins-flow-cytometry-isolated

Research

JoVE Journal

Biology

15.4K Views.  Université de Montréal, CRCHUM.  Qui descritto è un metodo per rilevare e quantificare proteine ​​di membrana mitocondriale esterna da immunomarcatura di mitocondri isolati da tessuto roditore e l'analisi mediante citometria a flusso. Questo metodo può essere esteso a valutare aspetti funzionali di sottopopolazioni mitocondriali. 

Video: Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

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

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

Misurazione quantitativa della traslocazione GLUT4 sulla membrana plasmatica mediante citometria di flusso

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

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 ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

L&#39;utilizzo di un robot per High-throughput cristallizzazione delle proteine ​​di membrana in mesofasi lipidici

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

Cristalli di raccolta e di Cryo-raffreddamento delle proteine ​​di membrana Grown in mesofasi lipidici per la determinazione della struttura di cristallografia macromolecolare

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

Multi-parametro di misura della apertura dei pori di transizione di permeabilità nel Cuore mouse mitocondri isolati

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

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 ...

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.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

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 ...

MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells

Mitochondria play a central role for cell metabolism, energy production and control of apoptosis. Inadequate mitochondrial function has been found responsible for very diverse diseases, ranging from neurological pathologies to cancer. Interestingly, mitochondria have recently been shown to display the capacity to be transferred between cell types, notably from human mesenchymal stem cells (MSC) to cancer cells in coculture conditions, with metabolic and functional consequences for the mitochondria recipient cells, further enhancing the current interest for the biological properties of these organelles.
Evaluating the effects of the transferred MSC mitochondria in the target cells is of primary importance to understand the biological outcome of such cell-cell interactions. The MitoCeption protocol described here allows the transfer of the mitochondria isolated beforehand from the donor cells to the target cells, using MSC mitochondria and glioblastoma stem cells (GSC) as a model system. This protocol has previously been used to transfer mitochondria, isolated from MSCs, to adherent MDA-MB-231 cancer cells. This mitochondria transfer protocol is adapted here for GSCs that present the specific particularity of growing as neurospheres in vitro. The transfer of the isolated mitochondria can be followed by fluorescence-activated cell sorting (FACS) and confocal imaging using mitochondria vital dyes. The use of mitochondria donor and target cells with distinct haplotypes (SNPs) also allows detection of the transferred mitochondria based on the concentration of their circular mitochondrial DNA (mtDNA) in the target cells. Once the protocol has been validated with these criteria, the cells harboring the transferred mitochondria can be further analyzed to determine the effects of the exogenous mitochondria on biological properties such as cell metabolism, plasticity, proliferation and response to therapy.

Here, a protocol (MitoCeption) is presented to transfer mitochondria, isolated from human mesenchymal stem cells (MSC), to glioblastoma stem cells (GSC), with the goal of studying their biological effects on GSC metabolism and functions. A similar protocol can be adapted to transfer mitochondria between other cell types.

MitoCeption: Trasferimento isolato umana MSC mitocondri di Glioblastoma cellule staminali

Mitochondria play a central role for cell metabolism, energy production and control of apoptosis. Inadequate mitochondrial function has been found ...

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 ...

Immunolocalizzazione della membrana esterna proteine ​​mediante citometria di flusso di mitocondri isolati

Immunolocalizzazione della membrana esterna proteine mediante citometria di flusso di mitocondri isolati