Questo lavoro è stato sostenuto dall&#39;Agenzia di ricerca sloveno con il progetto Z2-9229 e programma P2-0249. Questo video rappresenta il materiale integrativo per la &quot;Elettroporazione basato Tecnologie e Trattamenti&quot; laboratorio scientifico e corso post-laurea, organizzato ogni due anni dalla Facoltà di Ingegneria Elettrica presso l&#39;Università di Lubiana, Slovenia.

Parte I: passi preliminari <ol><li> In questo esperimento cinese linea di cellule ovariche di criceto (CHO-K1) è usato. Le cellule sono placcati in Lab-Tek II camere (2 pozzi, 4 cm 2 ciascuno) (Nalge Nunc, Germania) a ~ 0.7x10 5 cellule / ml in HAM-F12 terreno di coltura integrata con l&#39;8% di siero fetale bovino, 0,15 mg / ml L-glutamina, 16 mg / ml di gentamicina (tutti da Sigma-Aldrich, Steinheim, Germania), e 200 unità / ml Crystacillin (Pliva, Zagabria, Croazia), e incubate nel 5% CO 2 a 37 ° C. In alternativa, le cellule possono anche essere placcato in vetro scivola # 1 coperchio (0,13-0,16 mm di spessore), spalmato con un adesivo cellulari come polilisina. </li><li> Incubare le cellule nel loro terreno di coltura. L&#39;incubazione della durata di 2-4 ore le cellule che le rese sono ancora meno sferica, ma fermamente attaccato alla superficie con una piccola parte della loro membrana. In alternativa, dopo 16 a 20 ore di incubazione, le cellule sono completamente attaccati alla superficie e avere forme più complesse, ma la maggior parte di loro non sono ancora dividere. </li><li> Preparare una soluzione stock 10 mM di di-8-ANEPPS (Invitrogen, Eugene, Oregon, USA) con l&#39;aggiunta di 843 ml di DMSO (Sigma-Aldrich, Steinheim, Germania) a 5 mg del colorante nel flacone Invitrogen originale. La soluzione madre può essere conservato in frigorifero a 4 ° C per diversi mesi. Prima di iniziare gli esperimenti, scaldare la soluzione fino a quando i cristalli di DMSO sciogliere. </li><li> Alcune linee cellulari possono richiedere l&#39;uso di Pluronic per facilitare l&#39;inserimento colorante nella membrana cellulare. Pluronic possono essere acquistati nella soluzione madre al 20% in DMSO (F-127, Invitrogen, Eugene, Oregon, USA), o una soluzione madre della stessa concentrazione può essere preparata sciogliendo Pluronic in DMSO. Soluzione madre di Pluronic può essere conservato a temperatura ambiente. </li></ol> Parte II: Caricare le cellule con di-8-ANEPPS <ol><li> Mescolare 3 ml di 10 mM di-8-ANEPPS e 2,5 ml di 20% Pluronic in 1 ml della modifica Spinner (calcio-impoverito versione) del smem Medium minimo (medio M8167 o M4767, Sigma-Aldrich, Steinheim, Germania ) in una provetta Eppendorf da 1,5 ml. Questo produce una &quot;soluzione di caricamento&quot; contenente circa 30 micron di-8-ANEPPS e 0,05% del Pluronic. Per altri tipi di cellule, i loro terreni di coltura nativa può essere usato al posto di smem. </li><li> Sostituire il terreno di coltura nel laboratorio-Tek da camera con la soluzione di carico. Trasferimento della camera al frigorifero per 10 minuti a 4 ° C. A questa temperatura, l&#39;interiorizzazione del colorante attraverso la membrana plasmatica è in gran parte inibita. </li><li> Dopo la colorazione, lavare delicatamente il colorante in eccesso due o tre volte con smem puro. </li><li> Lascia 1,5 ml di smem nella camera. </li></ol> Parte III: Esperimento e acquisizione delle immagini <ol><li> Le cellule sono osservati utilizzando un microscopio a fluorescenza (nel nostro caso Zeiss Axiovert 200, Zeiss, Germania) dotato di un obiettivo ad immersione olio (x63, NA 1,4), un monocromatore (policroma IV, Visitron, Germania) ed una camera CCD raffreddata (VISICAM 1280, Visitron, Germania). Le immagini sono acquisite con MetaFluor 7.1.1 e trattati in MetaMorph 7.1.1 (sia Molecular Devices, Downingtown, PA, USA), ma altri software di acquisizione simili possono anche essere utilizzati. Impostare la lunghezza d&#39;onda di eccitazione nel software di acquisizione a 490 nm e scegliere un filtro adatto emissione per ANEPPS, per esempio, un filtro passa-banda centrata a 605 nm (605/55m, dicroiche 565 DCXR, Chroma, Rockingham, VT, USA). Se un monocromatore non è disponibile, un filtro di eccitazione centrato a 490 nm può essere utilizzato. </li><li> Posizionare la camera con le cellule sul palco microscopio, posizionare gli elettrodi sul fondo della camera e collegarli al generatore di impulsi. Il mezzo dovrebbe coprire gli elettrodi. </li><li> Per mantenere la vitalità cellulare e ridurre il riscaldamento, l&#39;impulso dovrebbe essere di ampiezza sufficientemente basso e breve durata. In questo esperimento un impulso quadrato con ampiezza 35 V e 50 ms di durata (sufficientemente basso per evitare elettroporazione) è generato con una tensione continua e una misura microprocessore dispositivo switcher. In alternativa, un generatore di impulsi commerciale, come 33210A (Agilent, Santa Clara, CA, USA) collegato ad un amplificatore o uno stimolatore commerciale, come S88 (Erba, West Warwick, Rhode Island, USA), può essere utilizzato. L&#39;impulso viene consegnato a due parallele Pt / Ir elettrodi a filo da 0,8 mm di diametro e 4 mm di distanza tra loro, creando un campo elettrico di circa 88 V / cm tra gli elettrodi e ΔΦ indurre. La distribuzione di campo esatta tra gli elettrodi utilizzati in questo esperimento è descritto altrove [1]. </li><li> Trova le cellule di interesse. Applicare un singolo impulso elettrico o una sequenza di impulsi. Per ogni impulso, di acquisire due immagini a fluorescenza: uno immediatamente prima dell&#39;impulso (l&#39;immagine di controllo), e uno durante l&#39;impulso (l&#39;immagine del polso). A causa della scarsa risposta di di-8-ANEPPS, i cambiamenti nella fluorescenza sono difficili da distinguere ad occhio nudo e diventano evidenti only dopo l&#39;elaborazione. La consegna degli impulsi deve essere sincronizzato con l&#39;acquisizione delle immagini, che è una caratteristica del software di acquisizione. Per evitare possibili fotodecolorazione e riscaldamento, l&#39;illuminazione può essere limitata alla durata dell&#39;impulso. </li></ol> Parte IV: L&#39;elaborazione delle immagini e analisi <ol><li> Aprire le immagini nel software MetaMorph. Per ogni impulso, sottrarre lo sfondo sia il controllo e l&#39;immagine di impulsi. </li><li> Scegliere una cella e impostare la regione di interesse in modo che corrisponda alla membrana. Misurare la fluorescenza lungo questa regione a immagine e di controllo degli impulsi e trasferire i valori di un foglio di calcolo. </li><li> Per ogni impulso, sottrarre i dati di controllo a partire dai dati di impulsi, e dividere il risultato per i dati di controllo per ottenere le relative variazioni di fluorescenza. Se una sequenza di impulsi viene applicata, i valori di fluorescenza relativi cambiamenti determinati per ciascun impulso può essere media per ottenere una misurazione più affidabile. </li><li> Trasformare le variazioni relative fluorescenza in ΔΦ utilizzando una curva di calibrazione. Una stima approssimativa di questa curva possono essere ottenute dalla letteratura, ma per una maggiore precisione, deve essere misurato per ciascuna configurazione particolare. Per le cellule e la configurazione utilizzata in questo esperimento una riduzione del 6% in fluorescenza corrisponde ad un aumento del 100 mV in ΔΦ [2]. </li><li> Infine, la trama la tensione in funzione della lunghezza dell&#39;arco relativo in un pacchetto di software grafici come Excel (Microsoft Corp., Redmond, WA, USA), Terreno Sigma (SYSTAT Software Inc., San Jose, CA, USA), o origine (OriginLab Corp., Northampton, MA, USA). La curva può anche essere lisciate con un filtro adatto, come ad esempio il filtro media mobile. </li></ol>

<ol>
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Eng. 34, 642-652 (2006).</li><li>Huang, C. J., Harootunian, A., Maher, M. P., Quan, C., Raj, C. D., McCormack, K., Numann, R., Negulescu, P. A., Gonzalez, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+voltage-gated+sodium-channel+blockers+by+electrical+stimulation+and+fluorescence+detection+of+membrane+potential.">Characterization of voltage-gated sodium-channel blockers by electrical stimulation and fluorescence detection of membrane potential.</a> Nat. Biotechnol. 24, 439-446 (2006).</li><li>Bedlack, R. S., Wei, M., Fox, S. H., Gross, E., Loew, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+electric+potentials+in+soma+and+neurite+membranes.">Distinct electric potentials in soma and neurite membranes.</a> Neuron. 13, 1187-1193 (1994).</li><li>Cheng, D. K. L., Tung, L., Sobie, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonuniform+responses+of+transmembrane+potential+during+electric+field+stimulation+of+single+cardiac+cells.">Nonuniform responses of transmembrane potential during electric field stimulation of single cardiac cells.</a> Am. J. Physiol. 277, H351-H362 (1999).</li><li>Teissie, J., Eynard, N., Gabriel, B., Rols, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electropermeabilization+of+cell+membranes.">Electropermeabilization of cell membranes.</a> Adv. Drug. Del. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+properties+of+tissue+and+cell+suspensions.">Electrical properties of tissue and cell suspensions.</a> Adv. Biol. Med. Phys. 5, 147-209 (1957).</li><li>Gowrishankar, T. R., Weaver, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+approach+to+electrical+modeling+of+single+and+multiple+cells.">An approach to electrical modeling of single and multiple cells.</a> Proc. Natl. Acad. Sci. USA. 100, 3203-3208 (2003).</li><li>Joshi, R. P., Hu, Q., Schoenbach, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+studies+of+cell+response+to+ultrashort,+high-intensity+electric+fields+-+Implications+for+intracellular+manipulation.">Modeling studies of cell response to ultrashort, high-intensity electric fields - Implications for intracellular manipulation.</a> IEEE Trans. Plasma Sci. 32, 1677-1686 (2004).</li><li>Gross, D., Loew, L. M., Webb, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+cell+membrane+potential+changes+induced+by+applied+electric+fields.">Optical imaging of cell membrane potential changes induced by applied electric fields.</a> Biophys. J. 50, 339-348 (1986).</li><li>Fluhler, E., Burnham, V. G., Loew, L. M. S. p. e. c. t. r. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=membrane+binding,+and+potentiometric+responses+of+new+charge+shift+probes.">membrane binding, and potentiometric responses of new charge shift probes.</a> Biochemistry. 24, 5749-5755 (1985).</li><li>Loew, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-sensitive+dyes:+measurement+of+membrane+potentials+induced+by+DC+and+AC+electric+fields.">Voltage-sensitive dyes: measurement of membrane potentials induced by DC and AC electric fields.</a> Bioelectromagnetics Suppl. 1, 179-189 (1992).</li><li>Hibino, M., Shigemori, M., Itoh, H., Nagayama, K., Kinosita, K. . J. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+conductance+of+an+electroporated+cell+analyzed+by+submicrosecond+imaging+of+transmembrane+potential.">Membrane conductance of an electroporated cell analyzed by submicrosecond imaging of transmembrane potential.</a> Biophys. J. 59, 209-220 (1991).</li><li>Lojewska, Z., Franks, D. L., Ehrenberg, B., Loew, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+effect+of+medium+and+membrane+conductance+on+the+amplitude+and+kinetics+of+membrane+potentials+induced+by+externally+applied+electric+fields.">Analysis of the effect of medium and membrane conductance on the amplitude and kinetics of membrane potentials induced by externally applied electric fields.</a> Biophys. J. 56, 121-128 (1989).</li><li>Zhang, J., Davidson, R. M., Wei, M. D., Loew, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+electric+properties+by+combined+patch+clamp+and+fluorescence+ratio+imaging+in+single+neurons.">Membrane electric properties by combined patch clamp and fluorescence ratio imaging in single neurons.</a> Biophys. J. 74, 48-53 (1998).</li><li>Montana, V., Farkas, D. L., Loew, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-wavelength+ratiometric+fluorescence+measurements+of+membrane-potential.">Dual-wavelength ratiometric fluorescence measurements of membrane-potential.</a> Biochemistry. 28, 4536-4539 (1989).</li><li>Knisley, S. B., Justice, R. K., Kong, W., Johnson, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ratiometry+of+transmembrane+voltage-sensitive+fluorescent+dye+emission+in+hearts.">Ratiometry of transmembrane voltage-sensitive fluorescent dye emission in hearts.</a> Am. J. Physiol. Heart Circ. Physiol. 279, H1421-H1433 (2000).</li><li>Pucihar, G., 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=A+time-dependent+numerical+model+of+transmembrane+voltage+inducement+and+electroporation+of+irregularly+shaped+cells.">A time-dependent numerical model of transmembrane voltage inducement and electroporation of irregularly shaped cells.</a> IEEE T. Biomed. Eng. 56, 1491-1501 (2009).</li></ol>

Misure della membrana indotta (transmembrana) tensione, ΔΦ, può essere importante in varie impostazioni sperimentali, come studi di canali voltaggio-dipendenti della membrana, la propagazione del potenziale d&#39;azione, la stimolazione delle cellule cardiache, o membrana elettroporazione di cellule [3, 4, 5, 6 , 7]. Con forme semplici cellule, ΔΦ può essere calcolato analiticamente. Ad esempio, per una cellula sferica, ΔΦ è data dalla equazione della Schwan s, in cui si afferma che la tensione è proporzionale alla forza del campo e le dimensioni delle cellule e segue la funzione coseno lungo la membrana [8, 9]. Per ulteriori forme cellulari complicati, ΔΦ possono discostarsi notevolmente dal coseno e deve essere determinato o in forma numerica, utilizzando un computer [2, 10, 11], o sperimentale, utilizzando un colorante potenziometrica [12, 13, 14, 15]. Uno dei coloranti potenziometrico ampiamente utilizzato per questo scopo è di-8-ANEPPS (di-8-butil-amino-naftil-etilene-piridinio-propil-solfonato), un colorante veloce con eccitazione e spettri di emissione dipende dal potenziale di membrana, non invasivo che permette osservazioni delle variazioni delle ΔΦ sulla membrana cellulare e di misurare il suo valore. In questo video, ci mostrano un approccio sperimentale per la determinazione di ΔΦ utilizzando di-8-ANEPPS. Il colorante è stato sviluppato dal professor Leslie Loew e colleghi [13, 14] presso l&#39;Università del Connecticut e appartiene alla classe di risposta rapida coloranti. di-8-ANEPPS è nonfluorescent in acqua e diventa fortemente fluorescenti quando si incorpora nel doppio strato lipidico della membrana cellulare. Un cambiamento nei risultati ΔΦ in un cambiamento della distribuzione di carica intramolecolare e corrispondenti cambiamenti nel profilo spettrale e l&#39;intensità di fluorescenza del colorante è. L&#39;intensità della fluorescenza di di-8-ANEPPS varia proporzionalmente alla variazione del ΔΦ, la risposta del colorante è lineare per tensioni che vanno da -280 mV a +250 mV [4, 16]. Cambiamenti relativamente piccoli nella fluorescenza del colorante, colorazione irregolare della membrana, e interiorizzazione colorante fare di-8-ANEPPS meno adatti per la misura assoluta del potenziale di membrana, ad esempio, la sua componente di riposo, anche se tali sforzi sono stati segnalati anche [17]. E &#39;, tuttavia, adatto per misurare grandi variazioni di tensione della membrana, come l&#39;insorgenza di potenziale di membrana indotta in cellule nonexcitable esposti a campi elettrici esterni [12, 13], o potenziali d&#39;azione nelle cellule eccitabili [4, 5]. Anche se non è qui applicata, di-8-ANEPPS permette anche la determinazione del ΔΦ da misurazioni raziometrico di eccitazione di fluorescenza [18] o di emissione [19], che aumenta la sensibilità della risposta e riduce gli effetti di cui sopra. Come di-8-ANEPPS macchie la membrana, che può essere utilizzato anche chiaramente come marker di membrana [2]. Uno degli svantaggi del colorante è che è incline a photobleaching, in modo che l&#39;esposizione prolungata alla luce forte dovrebbe essere evitato. Calibrazione del colorante viene eseguita con o (i) valinomicina ionoforo di potassio e un insieme di diverse concentrazioni di potassio in mezzo esterno [2,18], o (ii) patch-clamp in modalità morsetto di tensione [17]. Infine, con le misure di ΔΦ sulle cellule sferiche e le cellule di forme più complesse, il video dimostra l&#39;influenza della forma delle cellule sulla distribuzione spaziale di ampiezza e ΔΦ. Così per le cellule sferiche ΔΦ è vicino ad una coseno, in accordo con l&#39;equazione Schwan, mentre per ulteriori forme di cellule complicata la distribuzione spaziale della ΔΦ è più complessa [20] ...

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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>di-8-ANEPPS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>D-3167</td>
<td>potentiometric fluorescent dye</td>
</tr>
<tr>
<td>pluronic</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P3000MP</td>
<td>potassium ionophore</td>
</tr>
<tr>
<td>DMSO</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D2650</td>
<td>&nbsp;</td>
<tr>
<td>SMEM</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>M8167 or M4767</td>
<td>Spinner modification of the Minimum Essential Medium</td>
</tr>
<tr>
<td>Ham-F12</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>N4888</td>
<td>culture medium</td>
</tr>
<tr>
<td>fetal calf serum</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>F4135</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G7513</td>
<td>&nbsp;</td>
<tr>
<td>crystacillin</td>
<td>&nbsp;</td>
<td>Pliva</td>
<td>625110</td>
<td>antibiotic</td>
</tr>
<tr>
<td>gentamicin</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G1397</td>
<td>antibiotic</td>
</tr>
<tr>
<td>Lab-Tek II</td>
<td>&nbsp;</td>
<td>Nalge Nunc</td>
<td>155379</td>
<td>chamber</td>
</tr>
<tr>
<td>DC voltage supply</td>
<td>&nbsp;</td>
<td>Elektro-Automatik</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>microprocessor-controlled switcher</td>
<td>&nbsp;</td>
<td>Custom made</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>electrodes</td>
<td>&nbsp;</td>
<td>Custom made</td>
<td>&nbsp;</td>
<td>Pt/Ir</td>
</tr>		</tbody>
		</table>
		</div>

measuring the induced membrane voltage with di-8-anepps

Posizionamento di una cella in un campo elettrico esterno provoca un locale all&#39;interno di ridistribuzione della carica e al di fuori della cellula in prossimità della membrana cellulare, con una conseguente tensione attraverso la membrana. Questa tensione, chiamata tensione indotta membrana (tensione transmembrana anche indotta, o indotto differenza di potenziale transmembrana) e indicato con ΔΦ, esiste solo fino a quando il campo esterno. Se la tensione a riposo è presente sulla membrana, la tensione indotta sovrappone (aggiunge) su di esso. Usando uno dei coloranti fluorescenti potenziometrico, come di-8-ANEPPS, è possibile osservare le variazioni della ΔΦ sulla membrana cellulare e di misurare il suo valore non invasivo. di-8-ANEPPS diventa fluorescente quando fortemente legato al doppio strato lipidico della membrana cellulare, con il cambiamento delle intensità di fluorescenza proporzionale alla variazione di ΔΦ. Questo video mostra il protocollo per la misurazione ΔΦ utilizzo di-8-ANEPPS e dimostra anche l&#39;influenza della forma cella l&#39;ampiezza e la distribuzione spaziale della ΔΦ.

Watch this Scientific Journal Video about Measuring the Induced Membrane Voltage with Di-8-ANEPPS at JoVE.com

Measuring the Induced Membrane Voltage with Di-8-ANEPPS

Campo elettrico esterno induce una tensione sulla membrana di una cellula, chiamata potenziale di membrana indotta (ΔΦ). Usando il potenziometrica tintura di-8-ANEPPS, è possibile misurare la ΔΦ modo non invasivo. Questo video mostra il protocollo per la misurazione ΔΦ utilizzo di-8-ANEPPS.

Misurare la tensione indotta a membrana con Di-8-ANEPPS

Placement of a cell into an external electric field causes a local charge redistribution inside and outside of the cell in the vicinity of the cell ...

measuring-the-induced-membrane-voltage-with-di-8-anepps

Research

JoVE Journal

Biology

17.3K Views.  University of Ljubljana. Campo elettrico esterno induce una tensione sulla membrana di una cellula, chiamata potenziale di membrana indotta (ΔΦ). Usando il potenziometrica tintura di-8-ANEPPS, è possibile misurare la ΔΦ modo non invasivo. Questo video mostra il protocollo per la misurazione ΔΦ utilizzo di-8-ANEPPS.

Video: Measuring the Induced Membrane Voltage with Di-8-ANEPPS

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

Misura Vicino membrana plasmatica e Global Dynamics calcio intracellulare negli astrociti

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

Ricerca

Biologia

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

Misurazione delle proteine ​​plasmatiche Tariffe endocitico membrana da biotinilazione reversibile

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

Cristallizzare proteine ​​di membrana per la determinazione della struttura utilizzando Mesophases lipidica

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

Cristallizzazione delle proteine ​​di membrana lipidica in Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

Esaminando la dinamica conformazionale delle proteine ​​di membrana In situ Con il sito-diretta Etichettatura fluorescenza

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open Vaseline gap approach is used to obtain low noise recordings of ionic and gating currents from voltage-dependent ion channels expressed in Xenopus oocytes with high resolution of fast channel kinetics. With minor modification, voltage clamp fluorometry can be coupled to the cut-open oocyte protocol.

Il<em&gt; Xenopus</em&gt; Ovociti Cut-open vaselina Gap Tecnica Voltage-clamp Con fluorometria

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

Imaging Plasma Membrane Deformations With pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress.
Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or&#160;diD. DiD intercalates in the membrane plane with a &#34;fixed&#34; orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561&#160;nm laser (p-pol, s-pol). A 488&#160;nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

Imaging Plasma membrana Deformazioni Con pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

Measuring Glutathione-induced Feeding Response in Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

Misurazione glutatione indotta risposta alimentazione a Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

Microperfusione Tecnica per Indagare regolamento di microvasi permeabilità a Rat mesentere

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

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 ...