Questo lavoro è stato sostenuto dall&#39;Agenzia di ricerca sloveno (progetto J2-9764 e programma P2-0249). Questo video rappresenta materiale supplementare per il &quot;Elettroporazione basato Tecnologie e Trattamenti&quot; laboratorio scientifico e corso post-laurea, organizzato dalla Facoltà di Ingegneria Elettrica presso l&#39;Università di Lubiana, Slovenia.

I. Caricamento delle celle con celle inseguitori CMFDA e CMRA <ol><li> Gli esperimenti sono stati condotti su cellule precedentemente preparato delle linee cellulari di melanoma mouse (B16-F1). Le cellule sono coltivate in due separate 25 cm 2 fiasche cultura (TPP, ZDA) al 70-80% confluenza nel terreno di coltura DMEM (Dulbecco modificato medio di Eagle) supplementato con 10% siero fetale bovino, 0,15 mg / ml L-glutamina, 16 mg / ml di gentamicina (tutti da Sigma-Aldrich, Germania), 200 unità / ml crystacillin (Pliva, Croazia), e incubate nel 5% CO 2 a 37 ° C. </li><li> Preparare due soluzioni da 10 mM stock di inseguitori Cell (Invitrogen, USA) con l&#39;aggiunta di 10,76 microlitri e 9 microlitri (per il verde CMFDA e per Orange CMRA, rispettivamente) di DMSO (Sigma-Aldrich, Germania) a 50 mg del colorante in originale Invitrogen flaconcino. La soluzione madre può essere conservato in frigorifero a 4 ° C per pochi mesi. Prima di iniziare gli esperimenti, riscaldare la soluzione fino a quando i cristalli di DMSO sciogliere. </li><li> Preparare bicarbonato senza Krebs-tampone HEPES (130 mM NaCl, 4.7 mM KCl, 1,2 mM MgSO 4, 1,2 mM KH 2 PO 4, 11,7 mm D-glucosio, 1,3 mM CaCl 2, 10 HEPES mM, pH 7,4). In due tubi da 15 ml Eppendorf separatamente mix 2,1 ml di ciascuna soluzione madre (10 mM CMFDA o CMRA, rispettivamente) in 3 ml di bicarbonato senza Krebs-tampone HEPES. Questo produce una &quot;soluzione di carico&quot; che contiene circa 7 mM CMFDA (o CMRA). </li><li> Lavare le cellule due volte con il bicarbonato senza Krebs-tampone HEPES e quindi inserire il caricamento soluzioni nelle beute. Incubare le cellule per 30 minuti in 5% CO 2 a 37 ° C. Durante questo reagenti incubazione prima passare liberamente attraverso le membrane delle cellule, ma una volta all&#39;interno della cellula, i reagenti si trasformano in cellule impermeant prodotti di reazione fluorescente. </li><li> Dopo la prima incubazione, lavare e incubare cellule con terreno di coltura per altre due ore in 5% CO 2 a 37 ° C. </li><li> Trypsinize cellule in entrambi i flaconi (caricato con CMFDA e CMRA) e mescolare globuli rossi e verdi insieme in un rapporto di 1:1 in un tubo da centrifuga da 50 ml (TPP, ZDA). Regolare la concentrazione cellulare di 5 x 10 6 cellule / ml per diluizione con mezzo di DMEM o mediante concentrazione con centrifuga. Inserire una goccia 20 ml di sospensione di cellule in ciascun pozzetto della piastra 24 pozzetti (TPP, ZDA). Incubare cellule nel 5% CO 2 a 37 ° C per 20 minuti per consentire loro di attaccare un po &#39;alla superficie del pozzo e stabilire contatti cell. </li></ol> II. Elettrofusione <ol><li> Preparare isoosomolar tampone fosfato di potassio (10 mM KH2PO4, 10 mM K 2 HPO 4, 1mM MgCl2, 250 mM saccarosio) e hypoosmolar tampone fosfato di potassio (10 mM KH2PO4, 10 mM K 2 HPO 4, 1mM MgCl2, 75 mM saccarosio) . </li><li> Posizionare il pozzetti con le cellule al microscopio sul palco, la posizione degli elettrodi sul fondo del pozzo e collegarli al generatore di impulsi. </li><li> Lavare le cellule con 1 ml di isoosomolar tampone fosfato di potassio. Aggiungere 350 ml di hypoosmolar tampone fosfato di potassio al fine di indurre rigonfiamento delle cellule. Il buffer dovrebbe coprire gli elettrodi. </li><li> Lasciare le cellule nel buffer hypoosmolar per 2 minuti prima di applicare gli impulsi elettrici. Durante questo periodo afflusso di molecole d&#39;acqua all&#39;interno delle cellule a causa di squilibrio osmotico tra l&#39;interno e l&#39;esterno delle cellule causano un aumento del volume cellulare. Impulsi elettrici devono essere applicate quando le cellule sono vicini al loro volume massimo, prima di iniziare a regolamentare diminuire il volume. </li><li> Per raggiungere elettrofusione ottimale e mantenere la vitalità cellulare, i parametri ottimali di impulsi elettrici devono essere utilizzati. Questi dipendono da linee cellulari utilizzate [1]. In questo esperimento un treno di 8 impulsi rettangolari (ciascuno con durata di 100 ms a 1 Hz) viene applicato a ciascun campione, utilizzando un dispositivo elettroporazione (nel nostro caso Cliniporator, IGEA, Italia). Gli impulsi vengono consegnati a due parallele Pt / Ir elettrodi a filo da 0,8 mm di diametro e 5 mm di distanza tra loro, creando un campo elettrico di circa 1200 V / cm tra gli elettrodi in ogni bene, tranne per il controllo dei pozzi. </li><li> Lasciare le cellule indisturbato per 10 minuti dopo la consegna degli impulsi. Determinare il rendimento di fusione mediante microscopia a fluorescenza e contrasto di fase. </li></ol> III. Acquisizione di immagini e la determinazione del rendimento fusione <ol><li> Le cellule sono osservati utilizzando un microscopio a fluorescenza (nel nostro caso Zeiss Axiovert 200, Zeiss, Germania) equipaggiato con un obiettivo (x20) ed una camera CCD raffreddata (VISICAM 1280, Visitron, Germania). Le immagini vengono acquisite in MetaMorph 7.1.1 (Molecular Devices, USA), ma altri software di acquisizione simili possono anche essere utilizzati. CMFDA è eccitato con un monocromatore (policroma IV, Visitron, Germania) a 492 nm e CMRA a 548 nm. La fluorescenza della CMFDA e CMRA viene eseguita utilizzando due filtri di emissione, uno centrato a 535 nm (HQ535/30m, per CMFDA) e l&#39;altra centrata a 510 nm (D605/55m, per CMRA, sia Chroma, USA). L&#39;uso di specchi dicroici (Q515LP) hanno impedito la cross talk canale. </li><li> Acquisire tre immagini (contrasto di fase, fluorescenza rossa e verde) per cinque campi scelti a caso in ciascun pozzetto. Creare tre immagini per ogni canale tripletta immagine. In tale immagine citoplasma fluorescenti possono essere visti insieme con le membrane cellulari. Cellule fuse può quindi essere facilmente determinato [Figura 1]. </li><li> Contare tutti e tre i tipi di cellule (rosso, verde e fluorescente doppiamente) in ogni immagine a tre canali. Determinare la percentuale di cellule doppiamente fluorescenti dividendo il numero di cellule doppiamente fluorescenti con il numero di tutte le cellule in ogni immagine. Resa fusione è definita come la percentuale di cellule doppiamente fluorescenti moltiplicato per 2 in quanto la metà delle cellule fuse non vengono rilevati (quando le cellule del fusibile stesso colore). </li></ol> Rappresentante Risultati <img src="/files/ftp_upload/1991/1991fig1.jpg" alt="Figura 1" /> Figura 1 Tre immagine microscopia canale di B16F1 cellule dopo elettrofusione:. Contrasto di fase, CMRA fluorescenza (eccitazione a 548 nm) e CMFDA fluorescenza (eccitazione a 492 nm), ingrandimento obiettivo 20x

<ol>
	<li>&#268;emazar, M., Jarm, T., Miklav&#269;i&#269;, D., Ma&#269;ek-Lebar, A., Ihan, A., Kopitar, N. A., Ser&#353;a, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+electric-field+intensity+on+electropermeabilization+and+electrosensitivity+of+various+tumor-cell+lines+in+vitro.">Effect of electric-field intensity on electropermeabilization and electrosensitivity of various tumor-cell lines in vitro.</a> Electro. Magnetobiol. 17, 263-272 (1998).</li><li>Trontelj, K., Reber&#353;ek, M., Kandu&#353;er, M., &#269;urin &#353;erbec, V., Miklav&#269;i&#269;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+bulk+cell+electrofusion+in+vitro+for+production+of+human-mouse+heterohybridoma+cells.">Optimization of bulk cell electrofusion in vitro for production of human-mouse heterohybridoma cells.</a> Bioelectrochemistry. 74, 124-129 (2008).</li><li>Rols, M. -. P., Teissi&#233;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+electrically+induced+permeabilization+and+fusion+of+Chinese+hamster+ovary+cells+by+osmotic+pressure.">Modulation of electrically induced permeabilization and fusion of Chinese hamster ovary cells by osmotic pressure.</a> Biochemistry. 29, 4561-4567 (1990).</li><li>Neil, G., Zimmermann, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrofusion.">Electrofusion.</a> Methods in Enzymology. 220, 174-196 .</li><li>Abidor, I. G., Li, L. -. H., Hui, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+cell+pellets:+II.+Osmotic+properties,+electroporation,+and+related+phenomena:+Membrane+interactions.">Studies of cell pellets: II. Osmotic properties, electroporation, and related phenomena: Membrane interactions.</a> Biophysical journal. 67, 427-435 (1994).</li><li>Jaroszeski, M. J., Gilbert, R., Fallon, P. G., Heller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanically+facilitated+cell-cell+electrofusion.">Mechanically facilitated cell-cell electrofusion.</a> Biophys J. 67 (4), 1574-1581 (1994).</li><li>Ramos, C., Bonefant, D., Teissi&#233;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+hybridization+by+electrofusion+on+filters.">Cell hybridization by electrofusion on filters.</a> Analytical biochemistry. 302, 213-219 (2002).</li><li>Cao, Y., Yang, J., Yin, Z. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+high-throughput+cell+electrofusion+in+a+microelectrode-array+chip.">Study of high-throughput cell electrofusion in a microelectrode-array chip.</a> Microfluid Nanofluid. 5, 669-675 (2008).</li><li>U&#353;aj, M., Trontelj, M., Kandu&#353;er, M., Miklav&#269;i&#269;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+size+dynamics+and+viability+of+cells+exposed+to+hypotonic+treatment+and+electroporation+for+electrofusion+optimization.">Cell size dynamics and viability of cells exposed to hypotonic treatment and electroporation for electrofusion optimization.</a> Radiol. Oncol. 43, 108-119 (2009).</li><li>Teissi&#233;, J., Ramos, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+electric+field+pulse+induced+long-lived+permeabilization+and+fusogenicity+in+cell+membranes.">Correlation between electric field pulse induced long-lived permeabilization and fusogenicity in cell membranes.</a> Biophys. J. 74, 1889-1898 (1998).</li></ol>

Nessun conflitto di interessi dichiarati.

La capacità delle membrane cellulari di fondere in modo non specifico, ad esempio, da campi elettrici esterni, è importante per la biotecnologia, la medicina e la ricerca in biologia. Tale fusione non specifico permette la produzione di cellule ibride di grande valore e dei loro prodotti, come gli anticorpi monoclonali, e fornisce informazioni sui meccanismi fondamentali di fusione [2]. Elettrofusione è un metodo potenzialmente molto efficace in quanto può essere regolata correttamente a diversi tipi di cellule. Elettrofusione si ottiene quando le cellule in stretto contatto fisico sono messi a loro stato fusogenic (soggetto ad fusione) per mezzo di impulsi elettrici ad alta tensione. L&#39;efficienza di elettrofusione dipende da vari parametri che influiscono sulle due parti del processo di elettrofusione. La prima parte del processo di elettrofusione è il raggiungimento del contatto fisico tra le cellule, che possono essere ottenuti con metodi diversi [3-8]. Metodo di aderenza (cellule in crescita a confluenza) possono essere utilizzate in modo efficiente a causa di contatti cellule spontaneamente stabilimenti situati in zone di grandi dimensioni tra le cellule, tuttavia, che produce le cellule fuse molto grande con molti nuclei. Stiamo usando il metodo di aderenza modificato, dove celle più piccole (da 2 a 5 nuclei), che hanno maggiori probabilità di sopravvivere e proliferare, sono ottenuti (Figura 1). Il contatto tra le cellule anche beneficiare di rigonfiamento osmotico delle cellule, a causa di un trattamento osmotico utilizzati per l&#39;esperimento [9]. Seconda parte del processo di elettrofusione è il raggiungimento dello stato fusogenic delle membrane cellulari. Stato Fusogenic correla bene con lo stato electropermeabilized della membrana (cellule non sono specificamente permeabilizzate alle molecole che normalmente non può passare attraverso la membrana intatta) ed è regolato dagli stessi parametri degli impulsi elettrici (ampiezza, lunghezza, numero e frequenza) [10] . I valori dei parametri elettrici necessari per l&#39;elettroporazione ottimale [1] e elettrofusione differiscono tra cellule diverse e dipendono dalle dimensioni delle cellule e le loro proprietà biologiche. Parametri elettrici devono quindi essere ottimizzati per diverse linee cellulari, che vengono utilizzati come partner di fusione, per ottenere la fusione.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>CMRA</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>C34551</td>
<td>Cytosolic fluorescent dye</td>
</tr>
<tr>
<td>CMFDA</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>C7025</td>
<td>Cytosolic fluorescent dye</td>
</tr>
<tr>
<td>DMSO</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>D2650</td>
<td>&nbsp;</td>
<tr>
<td>DMEM </td>
<td>Reagent</td>
<td>Sigma-Aldrich </td>
<td>D5671</td>
<td>Dulbecco's modified Eagle's medium</td>
</tr>
<tr>
<td>Fetal calf serum</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>F4135</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>G7513</td>
<td>&nbsp;</td>
<tr>
<td>crystacillin</td>
<td>Reagent</td>
<td>Pliva</td>
<td>625110</td>
<td>antibiotic</td>
</tr>
<tr>
<td>gentamicin</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>G1397</td>
<td>antibiotic</td>
</tr>
<tr>
<td>Hepes</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>H0887</td>
<td>&nbsp;</td>
<tr>
<td>KH2PO4</td>
<td>Reagent</td>
<td>Merck </td>
<td>A124873 927</td>
<td>&nbsp;</td>
<tr>
<td>KH2PO4</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>4248</td>
<td>&nbsp;</td>
<tr>
<td>MgCl2</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>M-8266</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>Reagent</td>
<td>Fluka</td>
<td>71382</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>Reagent</td>
<td>Merck</td>
<td>A154336 908</td>
<td>&nbsp;</td>
<tr>
<td>MgSO4</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>M2643</td>
<td>&nbsp;</td>
<tr>
<td>D-glucose</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>G8270</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>C4901</td>
<td>&nbsp;</td>
<tr>
<td>sucrose</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>16104</td>
<td>&nbsp;</td>
<tr>
<td>Electric pulse generator</td>
<td>Tool</td>
<td>Igea</td>
<td>&nbsp;</td>
<td>Cliniporator VITAE</td>
</tr>
<tr>
<td>Multiwell plate</td>
<td>Tool</td>
<td>TPP</td>
<td>92424</td>
<td>&nbsp;</td>
<tr>
<td>50 ml centrifuge tube</td>
<td>Tool</td>
<td>TPP</td>
<td>91050</td>
<td>&nbsp;</td>
<tr>
<td>15 ml centrifuge tube</td>
<td>Tool</td>
<td>TPP</td>
<td>91015</td>
<td>&nbsp;</td>
<tr>
<td>25 cm2 culture flask</td>
<td>Tool</td>
<td>TPP</td>
<td>90026</td>
<td>&nbsp;</td>
<tr>
<td>Electrodes</td>
<td>Tool</td>
<td>Custom made</td>
<td>&nbsp;</td>
<td>Pt/Ir</td>
</tr>		</tbody>
		</table>
		</div>

cell electrofusion visualized with fluorescence microscopy

Elettrofusione di cellule è un metodo sicuro e non virale e non chimici che possono essere utilizzati per la preparazione di cellule ibride per la terapia umana. Elettrofusione consiste nell&#39;applicazione di brevi impulsi elettrici ad alta tensione per le cellule che sono in stretto contatto. Applicazione di breve, ad alta tensione impulsi elettrici provoca destabilizzazione della membrana plasmatica delle cellule. Membrane destabilizzato sono più permeabili di diverse molecole e anche incline alla fusione con le membrane vicine destabilizzato. Elettrofusione è quindi un metodo conveniente per raggiungere un non-specifico fusione di cellule molto diverse<em&gt; In vitro</em&gt;. Al fine di ottenere la fusione, le membrane cellulari, destabilizzato dal campo elettrico, deve essere in stretto contatto per consentire una fusione dei loro doppi strati lipidici e di conseguenza il loro citoplasma. In questo video, si dimostra efficiente elettrofusione di cellule<em&gt; In vitro</em&gt; Per mezzo del metodo di aderenza modificato. In questo modo, le cellule sono permesso di allegare solo leggermente la superficie del bene, in modo che media possono essere scambiati e le cellule conservano la loro forma sferica. Visualizzazione fusione è valutata la pre-etichettatura del citoplasma delle cellule con coloranti diversi di cellule fluorescenti inseguitore, la metà delle celle sono etichettate con arancia CMRA e l&#39;altra metà con verde CMFDA. Resa fusione è determinato dal numero di cellule doppiamente fluorescenti diviso con il numero di tutte le cellule moltiplicato per due.

Watch this Scientific Journal Video about Cell Electrofusion Visualized with Fluorescence Microscopy at JoVE.com

Cell Electrofusion Visualized with Fluorescence Microscopy

In questo video si dimostra efficiente elettrofusione di cellule<em&gt; In vitro</em&gt; Per mezzo di metodo rispetto modificati utilizzando elettroporazione e la rilevazione successiva visualizzazione fuso cellule con microscopia a fluorescenza.

Elettrosaldabili cellula visualizzate con microscopia a fluorescenza

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves ...

cell-electrofusion-visualized-with-fluorescence-microscopy

Research

JoVE Journal

Biology

14.0K Views.  University of Ljubljana. In questo video si dimostra efficiente elettrofusione di cellule In vitro Per mezzo di metodo rispetto modificati utilizzando elettroporazione e la rilevazione successiva visualizzazione fuso cellule con microscopia a fluorescenza.

Video: Cell Electrofusion Visualized with Fluorescence Microscopy

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

Visualizzazione singola molecola di DNA replica con microscopia a fluorescenza

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Ricerca

Biologia

Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and release from individual presynaptic terminals directly, we designed fluorescent false neurotransmitters as substrates for the synaptic vesicle monoamine transporter. Using these probes to image dopamine release in the striatum, we made several observations pertinent to synaptic plasticity. We found that the fraction of synaptic vesicles releasing neurotransmitter per stimulus was dependent on the stimulus frequency. A kinetically distinct "reserve" synaptic vesicle population was not observed under these experimental conditions. A frequency-dependent heterogeneity of presynaptic terminals was revealed that was dependent in part on D2 dopamine receptors, indicating a mechanism for frequency-dependent coding of presynaptic selection.

Hui Zhang and Niko G. Gubernator contributed equally to this work.

A new means to measure neurotransmission optically using fluorescent dopamine analogs.

Il rilascio di dopamina a singoli terminali presinaptici visualizzate con FFNs

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Time-lapse imaging di mitosi dopo la trasfezione di siRNA

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Basato sulla fluorescenza Misura Store-Entry operato di calcio in cellule vive: dal Cancer Cell coltura per fibra muscolare scheletrica

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

Quantitativa vivo cellulare-microscopio a fluorescenza Analisi di lievito Fission

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

Interazioni proteina-proteina visualizzati tramite bimolecular complementazione fluorescenza in protoplasti di tabacco e foglie

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

Preparazione del campione per microscopia a forza atomica Virion singola e imaging a fluorescenza a super risoluzione

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

Visualizzazione Frequenza Stromule con microscopia a fluorescenza

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.

Understanding the function of essential processes in bacteria is challenging. Fluorescence microscopy with target-specific dyes can provide key insights into microbial cell growth and cell cycle progression. Here, Agrobacterium tumefaciens is used as a model bacterium to highlight methods for live cell imaging for characterization of essential processes.

Vivere la microscopia di fluorescenza delle cellule per osservare i processi essenziali durante la crescita delle cellule microbiche

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of ...