Questo lavoro è stato sostenuto dal National Institutes of Health sovvenzioni AR055686 e AR060836 e borsa di studio postdottorato al dC da Francese Distrofia Muscolare (AFM). L&#39;impianto di imaging cellulare utilizzato in questo studio è sostenuto dal National Institutes of Health sovvenzioni HD040677.

1. Imaging Membrana cellulare Repair Uso Bulk (perle di vetro) Ferimento Questo protocollo permette separatamente segnando le cellule danneggiate e quelli che non riescono a guarire. Quantificare queste popolazioni di cellule richiede l&#39;utilizzo di tre condizioni: 1. Test (C1) - Le cellule possono riparare in presenza di Ca 2 +, 2. Controllo 1 (senza pregiudizio C2) - Le cellule vengono incubate in presenza di Ca 2 +, ma non feriti, e 3. Control 2 (senza C3 riparazione) - le cellule sono autorizzati a riparare in assenza di Ca 2 +. <ol><li> Crescere le cellule a&gt; 50% di confluenza in tre coprioggetto sterili e lavare C1 e C2 due volte con CIM a 37 ° C e C3 con PBS a 37 ° C e di trasferimento coprioggetto su O-ring in silicone. </li><li> Aggiungere 100 ml di soluzione di destrano FITC preriscaldata in CIM su C1 e C2 o C3 in PBS su. </li><li> Ferire membrana plasmatica in C1 e C3 da dolci 40 perle mg di vetro sopra il vetrino a temperatura ambiente mediante lamelle inclinabili manualmente indietro evia 6-8 volte con un angolo di 30 °. Nota: per lesioni lievi, assicurano le perle di vetro sono distribuite in modo uniforme, riducendo così al minimo le lesioni ripetute. Per migliorare la riproducibilità del pregiudizio tra i campioni, contemporaneamente effettuare la lesione branello di vetro dei diversi campioni da confrontare. </li><li> Evitare inoltre di rotolamento di perline, trasferire tutti i vetrini di un incubatore 37 ° C a CO 2 ambiente e consentire la riparazione di procedere per 5 min. </li><li> Senza lasciare le perle rotolano, rimuovere le perline di vetro e FITC destrano mediante lavaggio con CIM (C1 e C2) o PBS (C3) a 37 ° C. </li><li> Luogo coprioggetto torna sul ring e aggiungere 100 ml di soluzione di lisina TRITC destrano risolvibile preriscaldata in CIM su C1 e C2 o in PBS su C3. </li><li> Incubare a 37 ° C per 5 min a CO 2 ambiente. </li><li> Lavare coprioggetti due volte con preriscaldata CIM e fissare con PFA 4% per 10 min a RT. </li><li> Lavare due volte con PBS e incubare per 2 min a RT in Hoechst colorante. </li><li> Wash samples due volte con PBS, montare su un vetrino utilizzando supporti di montaggio e celle di immagine utilizzando un microscopio a epifluorescenza. </li><li> Utilizzare cellule da C2 per determinare il fondo rosso e verde intensità di colorazione e utilizzare questo valore di soglia per i canali rosso e verde per tutti i campioni (C1-C3). </li><li> Punteggio numero totale di cellule che sono a) verde (infortunato e riparato) e b), rosso o entrambi rosso e verde (infortunato, ma non è riuscito a riparare) in campioni C1 e C3. </li><li> Conte&gt; 100 cellule verdi per ogni condizione ed esprimere la frazione di cellule che non sono riusciti a riparare come percentuale di tutte le cellule danneggiate (verde e rosso). </li></ol> 2. Immagini dal vivo della cinetica di membrana cellulare di riparazione Following Laser Injury <ol><li> Lavare le cellule con preriscaldata CIM e poi mettere il vetrino in CIM con la tintura FM. </li><li> Posizionare il coprioggetto in un supporto in incubatrice all&#39;inizio stadio mantenuto a 37 ° C. </li><li> Selezionare una regione della membrana cellulare 1-2 mm 2, e per irradiare &lt;; 10 msec con il laser pulsato. Attenuare la potenza laser attraverso il software al 40-50% della potenza di picco. Potenza ottimale consente pregiudizio consistente, ma non letale e questo deve essere determinato per tentativi per ogni singolo strumento e linea cellulare utilizzato. </li><li> Per monitorare la riparazione, un&#39;immagine ogni 10 secondi in epifluorescenza e chiaro, iniziando prima di pregiudizio e proseguire per 3-5 minuti dopo la lesione. Nota: Per nessun controllo la riparazione, ripetere i passaggi 2,1-2,4 con le cellule feriti in PBS contenente colorante FM. </li><li> Per quantificare la cinetica di riparazione, misurare FM cellulare colorante fluorescente e tracciare la variazione di intensità (ΔF/F0) nel corso di imaging. Questi dati devono essere in media per oltre 10 celle in ogni stato e tracciata come media o il valore della singola cella, se necessario. </li></ol> 3. Imaging Bulk (perle di vetro) Infortunio Induced lisosomiale Esocitosi Gli esempi includono seguenti cellule cresciute a&gt; 50%confluenza: 1. Test (C1) - Cellule permesso di riparare in presenza di Ca 2 +, 2. Controllo 1 (C2; Nessun danno) - Cellule né feriti né incubate con l&#39;anticorpo primario, e 3. Control 2 (C3, n riparazione) - Cellule autorizzati a riparare in assenza di Ca 2 +. <ol><li> Lavare coprioggetto C1 e C2 due volte con CIM a 37 ° C e C3 con PBS a 37 ° C e trasferirli su O-ring in silicone. </li><li> Aggiungere 100 pl di preriscaldata lisina fissabile destrano TRITC in CIM su C1 e C2 e C3 in PBS su. </li><li> Ferire membrana plasmatica su C1 e C3 come al punto 1.3. </li><li> Evitare inoltre di rotolamento di perline, trasferire tutti i vetrini di un incubatore 37 ° C a CO 2 ambiente e consentire la riparazione di procedere per 5 min. </li><li> Rimuovere le perline di vetro e TRITC destrano lavando i coprioggetti in terreno di crescita freddo, assicurando ancora nessun rotolamento delle sfere di vetro sulle cellule. </li><li> Lavare i coprioggetti due volte con mezzo di crescita freddo e trasferire gli O-ring. </li><li> Per C1 e C3, Aggiungere 100 ml di ratto anticorpo anti LAMP1 topo in mezzi di crescita complete freddo e aggiungere il freddo, terreno di coltura completo C2. </li><li> Per consentire legame dell&#39;anticorpo lamelle incubare per 30 min a 4 ° C. </li><li> Lavare i coprioggetti tre volte con CIM freddo e fissare tutti i vetrini con il 4% PFA per 10 min a RT e poi risciacquare 3x con CIM. </li><li> Incubare tutte coprioggetto in 100 ml di soluzione di blocco per 15 minuti a RT </li><li> Incubare tutte coprioggetto in 100 microlitri Alexa Fluor 488 anticorpo anti ratto per 15 min a 4 ° C. </li><li> Lavare due volte con PBS e incubare per 2 minuti a temperatura ambiente in Hoechst. </li><li> Lavare le cellule due volte con PBS, montare su un vetrino utilizzando supporti di montaggio e immagine utilizzando un microscopio a epifluorescenza. </li><li> Utilizzo di immagini di cellule C2, determinare la colorazione di fondo aspecifica in rosso (TRITC destrano) e verde (Alexa Fluor 488 anticorpi) canali e usare questi valori colorazione di fondo di soglia i canali rosso e verde per tutti i campioni (C1-C3). &lt;/ Li&gt; <li> Utilizzare i rossi etichettati&gt; 100 cellule da C1 e C3 per misurare l&#39;intensità della colorazione LAMP1 (verde) in cellule danneggiate. Per un esperimento riuscito colorazione LAMP1 in celle da C3 sarà notevolmente inferiore rispetto alle cellule di C1. </li></ol> 4. Immagini dal vivo di Cell Membrane Injury Attivato Traffico subcellulare <ol><li> Fluorescently etichettare il vano di interesse trasfettando opportuno giornalista (es. CD63-GFP per lisosomi 8) oppure utilizzando coloranti fluorescenti 9. </li><li> Per i lisosomi etichettatura con colorante fluorescente, incubare cellule cresciute al 50% di confluenza in terreni di coltura contenente FITC-destrano </li><li> Lasciare destrano da endocitato per 2 ore (o più, se necessario per l&#39;etichettatura sufficiente endosomal con destrano dalla linea cellulare di interesse) in CO 2 incubatore. </li><li> Lavare le cellule con i media di crescita preriscaldate e incubare per 2 ore in un incubatore CO 2 per consentire a tuttiendocitosi destrano ad accumularsi nel lisosoma. </li><li> Prima di imaging, sciacquare il vetrino in preriscaldata CIM e montare in un supporto vetrino sul incubatore cima fase a 37 ° C. </li><li> Eseguire widefield (per il movimento in tutta la cellula) o TIRF (movimento alla superficie cellulare e esocitosi) immagini - le cellule che non hanno affollato FITC destrano lisosomi etichettati sono l&#39;ideale per l&#39;imaging dei movimenti e esocitosi dei singoli lisosomi. </li><li> Allineamento laser TIRF per microscopio di imaging: Usa 60X o 100X con obiettivo&gt; 1,45 NA. Impostare l&#39;angolo per incidente TIRF fascio laser usando l&#39;approccio del produttore. </li><li> Danneggiare la membrana cellulare irradiando una regione piccola (1-2 mm 2) per &lt;100 msec, con il laser pulsato a 40-50% attenuazione. </li><li> Per monitorare la risposta dei lisosomi alla cella di pregiudizio, celle di immagine a 4-6 fotogrammi / sec per almeno 2 minuti o più, a seconda delle dinamiche di esocitosi nelle cellule di interesse. </li></ol>

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Lesioni membrana cellulare in vivo verifica a causa di una varietà di stress fisiologici e diversi approcci sperimentali sono stati sviluppati per imitare questi. Questi includono il ferimento membrana cellulare delle cellule aderenti raschiando loro fuori il piatto o da passaging attraverso uno stretto 9,10 siringa foro. Seguendo tali lesioni guariscono le cellule in sospensione e non aderire alla matrice extracellulare, come avviene normalmente nel tessuto. Altri ancora, come l&#39;uso di pori for...

Membrana cellulare mantiene l&#39;integrità delle cellule, fornendo una barriera tra la cella e l&#39;ambiente extracellulare. Uno stimolo chimico, elettrico o meccanico che supera la normale soglia fisiologica così come la presenza di agenti patogeni invasori può ogni risultato lesioni alla membrana cellulare e innescare una risposta cellulare successiva per riparare questa lesione. Per sopravvivere queste lesioni alla membrana cellulare, cellule possiedono un meccanismo efficiente per la riparazione. Questo meccanismo è dipendente dal calcio e coinvolge traffico intracellulare di proteine ​​come MG53 annexins e tra gli altri, nonché compartimenti subcellulari come endosomi, lisosomi, Golgi vescicole derivate e mitocondri alla membrana cellulare feriti 1-7. Tuttavia, i dettagli della sequenza di eventi molecolari e subcellulari coinvolti nella riparazione membrana cellula danneggiata rimane poco compresa. Risposta di riparazione del cellulare può essere segregato in un orecchioly e risposte tardive. Le prime risposte, che si verificano in pochi secondi in scala minuti di tempo, sono estremamente importante nel determinare la natura delle risposte tardive che portano alla riparazione delle cellule successo o la morte delle cellule. Analisi del punto di arrivo sulla base di analisi biochimiche e cellulari di massa hanno contribuito a stabilire il coinvolgimento di processi molecolari e cellulari di riparazione. Ma, a causa della eterogeneità e rapidità di risposta di riparazione cellulare, saggi punto finale non riescono a fornire i dati cinetici e spaziali della sequenza di eventi che portano alla riparazione. Approcci che consentono pregiudizio controllata di membrana cellulare e permettono di monitorare la riparazione della membrana cellulare e subcellulare risposte associate ad alta risoluzione spaziale e temporale sono ideali per tali studi. Qui, tali approcci sono stati presentati. Due dei protocolli descrivono approcci per monitorare le reali cinetica momento della riparazione membrana cellulare e le risposte subcellulari associati al processo di riparazione in cellule vive seguenti inj laserury. Come complemento a queste analisi basate imaging cellulare dal vivo, saggi punto finale sono anche stati descritti che forniscono una misura basata sulla popolazione per il monitoraggio riparazione delle singole cellule e le risposte subcellulari associati. Per dimostrare la loro utilità questi approcci sono stati utilizzati per monitorare il traffico e l&#39;esocitosi dei lisosomi in risposta al danno membrana cellulare.

<table border="0" cellpadding="0" cellspacing="0" width="814" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>HBSS</td>
			<td>Sigma</td>
			<td>H1387</td>
			<td>Used to prepare CIM (HBSS/10 mM Hepes/2 mM Ca2+ pH 7.4)</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher</td>
			<td>BP410</td>
			<td>Used to prepare CIM (HBSS/10 mM Hepes/2 mM Ca2+ pH 7.4)</td>
		</tr>
		<tr>
			<td>FITC dextran (Lysine fixable)</td>
			<td>Invitrogen</td>
			<td>D1820</td>
			<td>2 mg/ml in CIM or PBS</td>
		</tr>
		<tr>
			<td>Glass beads</td>
			<td>Sigma</td>
			<td>G8772</td>
			<td></td>
		</tr>
		<tr>
			<td>TRITC dextran (Lysine fixable)</td>
			<td>Invitrogen</td>
			<td>D1818</td>
			<td>2 mg/ml in CIM or PBS</td>
		</tr>
		<tr>
			<td>PFA 16%</td>
			<td>EMS</td>
			<td>15710</td>
			<td>4% in PBS</td>
		</tr>
		<tr>
			<td>Hoechst</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td>1/10 000 in PBS</td>
		</tr>
		<tr>
			<td>FM dye</td>
			<td>Invitrogen</td>
			<td>T3163</td>
			<td>1 &#181;g/&#181;l in CIM or PBS</td>
		</tr>
		<tr>
			<td>FITC dextran</td>
			<td>Sigma</td>
			<td>FD40S</td>
			<td>2 mg/ml in growth medium</td>
		</tr>
		<tr>
			<td>LAMP1&#160;</td>
			<td>Santa Cruz</td>
			<td>sc-19992</td>
			<td>1/300 in growth medium</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 chicken anti-rat IgG</td>
			<td>Invitrogen</td>
			<td>A21470</td>
			<td>1/500 in blocking solution</td>
		</tr>
		<tr>
			<td>Mounting media</td>
			<td>Dako</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Fisher</td>
			<td>12-545-86</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottom petridish</td>
			<td>MatTek corporation</td>
			<td>P35G-1.0-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone O ring</td>
			<td>Bellco</td>
			<td>1943-33315</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip holder</td>
			<td>Bellco</td>
			<td>1943-11111</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen</td>
			<td>A-7816</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>VWR</td>
			<td>12001-566</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>VWR</td>
			<td>MP1400-500H</td>
			<td>Used for growth medium: 10% FBS, 1% P/S in DMEM</td>
		</tr>
		<tr>
			<td>Penicillin/Sreptomycin</td>
			<td>VWR</td>
			<td>12001-350</td>
			<td>Used for growth medium: 10% FBS, 1% P/S in DMEM</td>
		</tr>
		<tr>
			<td>Chicken serum</td>
			<td>Sigma</td>
			<td>C5405</td>
			<td>5% chicken serum in CIM is used for blocking solution during immunostaining</td>
		</tr>
		<tr>
			<td>PBS (Ca2+ and Mg2+ free)</td>
			<td>VWR</td>
			<td>12001-664</td>
			<td></td>
		</tr>
		<tr>
			<td>Epifluorescence microscope</td>
			<td>Olympus America, PA</td>
			<td>IX81</td>
			<td></td>
		</tr>
		<tr>
			<td>TIRF illuminator&#160;</td>
			<td>Olympus America, PA</td>
			<td>Cell^TIRF (IEC60825-1:2007)</td>
			<td></td>
		</tr>
		<tr>
			<td>Epifluorescence illuminator</td>
			<td>Sutter Instruments, Novato CA</td>
			<td>Lambda DG-4 (DG-4 30)</td>
			<td></td>
		</tr>
		<tr>
			<td>CCD Camera</td>
			<td>Photometrics, Tucson, AZ</td>
			<td>Evolve 512 EMCCD&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Image acquisition and anaysis software</td>
			<td>Intelligent Imaging Innovations, Inc. Denver, CO</td>
			<td>Slidebook 5</td>
			<td></td>
		</tr>
		<tr>
			<td>Pulsed laser</td>
			<td>Intelligent Imaging Innovations, Inc. Denver, CO</td>
			<td>Ablate (3iL13)</td>
			<td></td>
		</tr>
		<tr>
			<td>Stage top incubator</td>
			<td>Tokaihit, Japan</td>
			<td>INUBG2ASFP-ZILCS</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging cell membrane injury and subcellular processes involved in repair

La capacità delle cellule danneggiate per guarire è un processo cellulare fondamentale, ma i meccanismi cellulari e molecolari coinvolti nella guarigione delle cellule feriti sono poco conosciute. Qui saggi sono descritti per monitorare la capacità e la cinetica di guarigione delle cellule in coltura dopo la lesione localizzata. Il primo protocollo descrive un approccio basato punto di fine di valutare simultaneamente cellule riparazione della membrana capacità di centinaia di cellule. Il secondo protocollo descrive un metodo di immagini in tempo reale per monitorare la cinetica di riparazione membrana cellulare in cellule singole seguenti lesioni localizzate con un laser pulsato. Come cellule di guarigione feriti comporta tratta di specifiche proteine ​​e dei compartimenti subcellulari al sito della lesione, il terzo protocollo descrive l&#39;uso di approccio basato punto finale di cui sopra per valutare uno di questi eventi traffico (esocitosi lisosomiale) in centinaia di cellule danneggiate simultaneamente e l&#39;ultimo protocollo descrive l&#39;uso di lesioni laser pulsato con micros TIRFcopia di monitorare le dinamiche dei singoli compartimenti subcellulari in cellule danneggiate ad alta risoluzione spaziale e temporale. Mentre i protocolli che seguono descrivono l&#39;uso di questi approcci per studiare il legame tra riparazione membrana cellulare e lisosomiale esocitosi in cellule muscolari coltivate, possono essere applicate come tali per qualsiasi altra cellula in coltura aderente e compartimento subcellulare di scelta.

Protocolli descritti qui per l&#39;imaging singola cellula sono di monitorare la capacità e la cinetica di riparazione della membrana cellulare (protocolli 1 e 2) e il traffico subcellulare e la fusione dei lisosomi durante la riparazione (protocolli 3 e 4). Protocollo 1 mostra un saggio di massa che permette la marcatura tutte le cellule danneggiate e individuare le cellule danneggiate che non sono riusciti a riparare. I risultati in figura 1 mostrano che mentre le cellule...

Watch this Scientific Journal Video about Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair at JoVE.com

Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

Il processo di guarigione cellule feriti comporta tratta di specifiche proteine ​​e dei compartimenti subcellulari al sito della lesione membrana cellulare. Questo protocollo descrive saggi per monitorare questi processi.

Imaging Membrana cellulare Pregiudizio e processi subcellulari coinvolti nella riparazione

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly ...

imaging-cell-membrane-injury-subcellular-processes-involved

Research

JoVE Journal

Biology

14.9K Views.  Children's National Medical Center.  Il processo di guarigione cellule feriti comporta tratta di specifiche proteine ​​e dei compartimenti subcellulari al sito della lesione membrana cellulare. Questo protocollo descrive saggi per monitorare questi processi. 

Video: Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many degenerative human diseases. The recent discovery of MG53 as a key component of the membrane resealing machinery allows for a better molecular understanding of the basic biology of tissue repair, as well as for potential translational applications in regenerative medicine. Here we detail the experimental protocols for exploring the in vivo function of MG53 in repair of muscle injury using treadmill exercise protocols on mouse models, for testing the ex vivo membrane repair capacity by measuring dye entry into isolated muscle fibers, and for monitoring the dynamic process of MG53-mediated vesicle trafficking and cell membrane repair in cultured cells using live cell confocal microscopy.

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Described here are protocols used to visualize the dynamic process of MG53-mediated cell membrane repair in whole animals and at the cellular level. These methods can be applied to investigate the cell biology of plasma membrane resealing and regenerative medicine.

Visualizzazione di MG53-mediata di riparazione della membrana cellulare Utilizzando In vivo E In vitro Sistemi

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many ...

Ricerca

Biologia

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth.
The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.

This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.

Rat Esteriorizzazione mesentere: un modello per indagare le dinamiche cellulari coinvolti nell&#39;angiogenesi

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease ...

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event that triggers the repair of mechanical wounds on the plasma membrane within ~30 sec. Recent studies revealed that mammalian cells also reseal their plasma membrane after permeabilization with pore forming toxins in a Ca2+-dependent process that involves exocytosis of the lysosomal enzyme acid sphingomyelinase followed by pore endocytosis. Here, we describe the methodology used to demonstrate that the resealing of cells permeabilized by the toxin streptolysin O is also rapid and dependent on Ca2+ influx. The assay design allows synchronization of the injury event and a precise kinetic measurement of the ability of cells to restore plasma membrane integrity by imaging and quantifying the extent by which the liphophilic dye FM1-43 reaches intracellular membranes. This live assay also allows a sensitive assessment of the ability of exogenously added soluble factors such as sphingomyelinase to inhibit FM1-43 influx, reflecting the ability of cells to repair their plasma membrane. This assay allowed us to show for the first time that sphingomyelinase acts downstream of Ca2+-dependent exocytosis, since extracellular addition of the enzyme promotes resealing of cells permeabilized in the absence of Ca2+.

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Live imaging of cells exposed to the lipophilic dye FM1-43 allows precise determination of the kinetics by which pore-forming toxins are removed from the plasma membrane. This is a sensitive assay that can be used to assess requirements for Ca2+, sphingomyelinase and other factors on plasma membrane repair.

Vivere Imaging test per la valutazione dei ruoli di Ca<sup&gt; 2 +</sup&gt; E sfingomielinasi nella riparazione di Pore-formano ferite Tossina

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Intravitale Microscopia per le strutture Imaging subcellulari in topi vivi Esprimere proteine ​​fluorescenti

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Ground State Depletion Super-resolution Imaging in Mammalian Cells

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to dramatic leaps in our understanding of how cells function. The development and availability of super-resolution microscopy has considerably extended the limits of optical resolution from ~250-20 nm. Biologists are no longer limited to describing molecular interactions in terms of colocalization within a diffraction limited area, rather it is now possible to visualize the dynamic interactions of individual molecules. Here, we outline a protocol for the visualization and quantification of cellular proteins by ground-state depletion microscopy&#160;for fixed cell imaging. We provide examples from two different membrane proteins, an element of the endoplasmic reticulum translocon, sec61&#946;, and a plasma membrane-localized voltage-gated L-type Ca2+ channel (CaV1.2). Discussed are the specific microscope parameters, fixation methods, photo-switching buffer formulation, and pitfalls and challenges of image processing.

This article outlines a protocol for the detection of one or more plasma and/or intracellular membrane proteins using Ground State Depletion (GSD) super-resolution microscopy in mammalian cells. Here, we discuss the benefits and considerations of using such approaches for the visualization and quantification of cellular proteins.

Stato fondamentale lo svuotamento super-resolution Imaging in cellule di mammifero

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to ...

Super-Resolution Live Cell Imaging of Subcellular Structures

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally been restricted to be used only on fixed samples or live cells outside of tissue labeled with strong fluorescent signal. Current super-resolution live cell imaging techniques require the use of special fluorescence probes, high illumination, multiple image acquisitions with post-acquisition processing, or often a combination of these processes. These prerequisites significantly limit the biological samples and contexts that this technique can be applied to.
Here we describe a method to perform super-resolution (~140 nm XY-resolution) time-lapse fluorescence live cell imaging in situ. This technique is also compatible with low fluorescent intensity, for example, EGFP or mCherry endogenously tagged at lowly expressed genes. As a proof-of-principle, we have used this method to visualize multiple subcellular structures in the Drosophila testis. During tissue preparation, both the cellular structure and tissue morphology are maintained within the dissected testis. Here, we use this technique to image microtubule dynamics, the interactions between microtubules and the nuclear membrane, as well as the attachment of microtubules to centromeres.
This technique requires special procedures in sample preparation, sample mounting and immobilizing of specimens. Additionally, the specimens must be maintained for several hours after dissection without compromising cellular function and activity. While we have optimized the conditions for live super-resolution imaging specifically in Drosophila male germline stem cells (GSCs) and progenitor germ cells in dissected testis tissue, this technique is broadly applicable to a variety of different cell types. The ability to observe cells under their physiological conditions without sacrificing either spatial or temporal resolution will serve as an invaluable tool to researchers seeking to address crucial questions in cell biology.

Presented here is a protocol for super-resolution live-cell imaging in intact tissue. We have standardized the conditions for imaging a highly sensitive adult stem cell population in its native tissue environment. This technique involves balancing temporal and spatial resolution to allow for the direct observation of biological phenomena in live tissue.

Imaging di cellule vive super-risoluzione di strutture subcellulari

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally ...

Photodiode-Based Optical Imaging for Recording Network Dynamics with Single-Neuron Resolution in Non-Transgenic Invertebrates

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light represents a revolutionary advance for studies of the neural basis of behavior. However, a downside of this development is its tendency to focus investigators on a very small number of &#8220;designer&#8221; organisms (e.g., C. elegans and Drosophila), potentially negatively impacting the pursuit of comparative studies across many species, which is needed for identifying general principles of network function. The present article illustrates how optical recording with voltage-sensitive dyes in the brains of non-transgenic gastropod species can be used to rapidly (i.e., within the time course of single experiments) reveal features of the functional organization of their neural networks with single-cell resolution. We outline in detail the dissection, staining, and recording methods used by our laboratory to obtain action potential traces from dozens to ~150 neurons during behaviorally relevant motor programs in the CNS of multiple gastropod species, including one new to neuroscience &#8211; the nudibranch Berghia stephanieae. Imaging is performed with absorbance voltage-sensitive dyes and a 464-element photodiode array that samples at 1,600 frames/second, fast enough to capture all action potentials generated by the recorded neurons. Multiple several-minute recordings can be obtained per preparation with little to no signal bleaching or phototoxicity. The raw optical data collected through the methods described can subsequently be analyzed through a variety of illustrated methods. Our optical recording approach can be readily used to probe network activity in a variety of non-transgenic species, making it well suited for comparative studies of how brains generate behavior.

This protocol presents a method for imaging neuronal population activity with single-cell resolution in non-transgenic invertebrate species using absorbance voltage-sensitive dyes and a photodiode array. This approach enables a rapid workflow, wherein imaging and analysis can be pursued over the course of a single day.

Imaging ottico basato su fotodiodi per la registrazione di dinamiche di rete con risoluzione a singolo neurone in invertebrati non transgenici

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light ...

Isolating and Imaging Live, Intact Pacemaker Regions of Mouse Renal Pelvis by Vibratome Sectioning

The renal pelvis (RP) is a funnel-shaped, smooth muscle structure that facilitates normal urine transport from the kidney to the ureter by regular, propulsive contractions. Regular RP contractions rely on pacemaker activity, which originates from the most proximal region of the RP at the pelvis-kidney junction (PKJ). Due to the difficulty in accessing and preserving intact preparations of the PKJ, most investigations on RP pacemaking have focused on single-cell electrophysiology and Ca2+ imaging experiments. Although important revelations on RP pacemaking have emerged from such work, these experiments have several intrinsic limitations, including the inability to accurately determine cellular identity in mixed suspensions and the lack of in situ imaging of RP pacemaker activity. These factors have resulted in a limited understanding of the mechanisms that underlie normal rhythmic RP contractions. In this paper, a protocol is described to prepare intact segments of mouse PKJ using a vibratome sectioning technique. By combining this approach with mice expressing cell-specific reporters and genetically encoded Ca2+ indicators, investigators may be able to more accurately study the specific cell types and mechanisms responsible for peristaltic RP contractions that are vital for normal urine transport.

The goal of this protocol is to isolate intact pacemaker regions of the mouse renal pelvis using vibratome sectioning. These sections can then be used for in situ Ca2+ imaging to elucidate Ca2+ transient properties of pacemaker cells and other interstitial cells in vibratome slices.

Isolamento e imaging delle regioni pacemaker intatte e intatte della pelvi renale del topo mediante sezionamento del vibratoma

The renal pelvis (RP) is a funnel-shaped, smooth muscle structure that facilitates normal urine transport from the kidney to the ureter by regular, ...

Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the zebrafish cardiovascular tissues have been limiting in studying their electrophysiological properties. Previous methods have been described for dissection of zebrafish hearts and isolation of ventricular cardiac myocytes. However, the isolation of zebrafish atrial and vascular myocytes for electrophysiological characterization was not detailed. This work describes new and modified enzymatic protocols that routinely provide isolated juvenile and adult zebrafish ventricular and atrial cardiomyocytes, as well as vascular smooth muscle (VSM) cells from the bulbous arteriosus, suitable for patch-clamp experiments. There has been no literary evidence of electrophysiological studies on zebrafish cardiovascular tissues isolated at embryonic and larval stages of development. Partial dissociation techniques that allow patch-clamp experiments on individual cells from larval and embryonic hearts are demonstrated.

The present protocol describes the acute isolation of viable cardiac and vascular smooth muscle cells from adult, juvenile, larval, and embryonic zebrafish (Danio rerio), suitable for electrophysiological studies.

Isolamento di cellule muscolari lisce cardiache e vascolari da pesci zebra adulti, giovanili, larvali ed embrionali per studi elettrofisiologici

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the ...

Imaging Neutrophil Migration in the Mouse Skin to Investigate Subcellular Membrane Remodeling Under Physiological Conditions

The study of immune cell recruitment and function in tissues has been a very active field over the last two decades. Neutrophils are among the first immune cells to reach the site of inflammation and to participate in the innate immune response during infection or tissue damage. So far, neutrophil migration has been successfully visualized using various in vitro experimental systems based on uniform stimulation, or confined migration under agarose, or micro-fluidic channels. However, these models do not recapitulate the complex microenvironment that neutrophils encounter in vivo. The development of multiphoton microscopy (MPM)-based techniques, such as intravital subcellular microscopy (ISMic), offer a unique tool to visualize and investigate neutrophil dynamics at subcellular resolutions under physiological conditions. In particular, the ear of a live anesthetized mouse provides an experimental advantage to follow neutrophil interstitial migration in real-time due to its ease of accessibility and lack of surgical exposure. ISMic provides the optical resolution, speed, and depth of acquisition necessary to track both cellular and, more importantly, subcellular processes in 3D over time (4D). Moreover, multi-modal imaging of the interstitial microenvironment (i.e., blood vessels, resident cells, extracellular matrix) can be readily accomplished using a combination of transgenic mice expressing select fluorescent markers, exogenous labeling via fluorescent probes, tissue intrinsic fluorescence, and second/third harmonic generated signals. This protocol describes 1) the preparation of neutrophils for adoptive transfer into the mouse ear, 2) different settings for optimal sub-cellular imaging, 3) strategies to minimize motion artifacts while maintaining a physiological response, 4) examples of membrane remodeling observed in neutrophils using ISMic, and 5) a workflow for the quantitative analysis of membrane remodeling in migrating neutrophils in vivo.

Neutrophil migration relies on the rapid and continuous remodeling of the plasma membrane in response to chemoattractant and its interactions with the extracellular microenvironment. Described herein is a procedure based on Intravital Subcellular Microscopy to investigate the dynamic of membrane remodeling in neutrophils injected in the ear of anesthetized mice.

Imaging della migrazione dei neutrofili nella pelle del topo per studiare il rimodellamento della membrana subcellulare in condizioni fisiologiche

The study of immune cell recruitment and function in tissues has been a very active field over the last two decades. Neutrophils are among the first ...