D. Il Sames grazie Harold G. &amp; Leila Y. Mathers Charitable Foundation e della Columbia University Iniziative in Scienze e Ingegneria. D. Sames e D. Sulzer ringraziare la Fondazione McKnight per le innovazioni tecnologiche nel McKnight Premio Neuroscienze D. Sulzer grazie NIDA, NIMH, e il Picower e Fondazioni Malattia di Parkinson. H. grazie Zhang NARSAD. RH Edwards ringrazia la Michael J. Fox Foundation, la National Parkinson Foundation, NIDA e NIMH. Ringraziamo Robert Burke per 6-OHDA iniezioni e consulenza, Sonders Marchio di discussione utile, Merek Siu per la programmazione di analisi delle immagini, e Jan Schmoranzer per l&#39;assistenza tecnica con l&#39;installazione di microscopia TIRF.

Questo metodo è stato utilizzato nella ricerca riportata in <a href="http://www.sciencemag.org/cgi/content/full/324/5933/1441">gubernator et al. Science 324 (5933). 1441-1444 (2009)</a> . 1. Preparazione acuta fette striatale <ol><li> Prima di preparare acuta fettine striatali, si deve ossigenare il liquido cerebrospinale artificiale (ASCF) e raffreddare con ghiaccio per almeno 15 minuti prima l&#39;estrazione del cervello. Tenere il freddo ghiaccio ACSF e ossigenato durante la dissezione. ACSF (in mM): NaCl 125, KCl 2.5, NaHCO 3 26, CaCl 2 2,4, MgSO4 1.3, KH 2 PO 4 0,3, glucosio 10, HEPES 5, pH 7,3-7,4, 290-295 mOsm. </li><li> Decapitare topo maschio senza anestesia. </li><li> Estrarre l&#39;intero cervello dal mouse, e montarlo sul vassoio vibratome immessi sul vibratome con Krazy Glue ®. Estrazione del cervello e di montaggio deve essere fatto entro 2 minuti. Durante questo tempo, assicurarsi di mantenere il freddo ghiaccio ACSF e ossigenato per mantenere il tessuto sano. </li><li> Tagliate le fette coronali del cervello striatale a spessore 250μm tra bregma 1,54-0,62 1. Tre fette si ottiene che possono poi essere tagliato in 6 fette di mezzo con un ago. </li><li> Incubare le fette di cervello in ACSF ossigenata a temperatura ambiente per almeno 1 ora prima del caricamento della sonda. Fette di buon display FFN511 carico e rimane attivo per le immagini fino a 4 ore dopo la preparazione fetta. </li></ol> 2. Caricamento FFN511 <ol><li> Preparare FFN511 soluzione di carico (10μM FFN511 in ACSF); anche preparare 100μM ADVASEP-7 in ACSF. Tutte le soluzioni devono essere preparate di fresco e ossigenato almeno 15 minuti prima di caricare in fette. </li><li> Individualmente incubare fetta con FFN511 soluzione di carico per 30 minuti a temperatura ambiente. </li><li> Quindi rimuovere il colorante legato al tessuto extracellulare incubando la fetta caricata in ossigenato 100μM ADVASEP-7 ACSF per 30 minuti a temperatura ambiente 2. Fette sono ora pronti per l&#39;imaging. </li></ol> 3. Imaging FFN511 in fettine di cervello Imaging di FFN511 nel fette viene eseguita utilizzando microscopio a scansione laser multiphoton. Stiamo usando una prateria di Ultima multiphoton microscopio a scansione laser (Stavamo usando un obiettivo Zeiss LSM 510 NLO multiphoton microscopio a scansione laser in precedenza e alcuni risultati sono stati ottenuti con il microscopio Zeiss). <ol><li> Posizionare il FFN511-caricato fetta nella camera di registrazione (RC-27L, Warner) e con superfuse ACSF ossigenato ad un flusso di 1-2 ml al minuto. Poi posto l&#39;arpa platino con corde di nylon sulla parte superiore della fetta per minimizzare i movimenti durante l&#39;esperimento. Per ridurre ulteriormente il movimento, permettono la fetta si stabilizzi per almeno 10 minuti nella camera prima di imaging. </li><li> Visualizzare e localizzare il corpo striato dorsale con luminescenza in campo chiaro con un obiettivo 10x immersione in acqua. </li><li> Posto e la posizione degli elettrodi di tungsteno bipolare intrecciato in questa regione se si esegue un esperimento di stimolazione. L&#39;area di imaging ideale si trova nel raggio di 300 micron tra le due punte degli elettrodi bipolari stimolante. Posto questa regione al centro del campo visivo. </li><li> Immagine FFN511 marcato terminali striatali sotto (0,9 NA) obiettivo ultravioletti 63x immersione in acqua. FFN511 è eccitato a 760 nm con un laser da Mai Tai Fisica spettrale e fluorescenza ottimale di terminali striatali è visto attraverso un filtro passa banda (480-520 nm). Le immagini vengono catturate in 12-bit con formato 75 x 75 micron regioni di interesse a 512 x 512 pixel di risoluzione. Per stimolato esperimenti decolorazione, per compensare z turno, una serie z di immagini 5-7, separati da 1 micron nel piano z, si ottiene per ogni periodo di tempo. </li></ol> 4. FFN511 decolorante Il FFN511 etichetta può essere decolorato sia un&#39;alta concentrazione di KCl (usiamo 70 mm KCl), (+)-amfetamina solfato (AMPH, 20 mM), o la stimolazione elettrica. Nell&#39;esperimento decolorazione KCl, quando una soluzione ad alta ACSF potassio viene applicata la fetta cervello, il FFN511 destains etichetta entro 2 minuti. Record xyz-t immagini per tracciare FFN511 decolorazione nel tempo. Di seguito sono descritte stimolazione elettrica-dipendente decolorazione terminale della dopamina: <ol><li> Per tenere traccia FFN511 decolorazione nel tempo, xyz-t immagini vengono registrate. Per garantire minimo movimento della fetta (meno di 4 micron di z) e non photobleaching dai, immagini in tempo laser serie il controllo della z-stack dovrebbe essere ottenuto per almeno 5 minuti prima della stimolazione. In caso contrario, regolare la potenza del laser. </li><li> A seguito di queste immagini di controllo, continuare la xyz-t immagini mentre inizio la stimolazione a frequenza di 1, 4 o 20 Hz. Gli stimoli a 1, 4 o 20 Hz (300 ms x 1 mA) vengono applicati al corpo striato a livello locale da parte di un Iso-Flex isolatore stimolo innescato da un Maestro-8 generatore di impulsi (AMPI, Gerusalemme, Israele) con elettrodi bipolari. Per ridurre al minimola variazione della depolarizzazione dei siti di rilascio, abbiamo utilizzato un protocollo di stimolazione applicare il 150% della massima intensità di stimolazione determinata dalla voltammetria ciclica. </li><li> Utilizzare il software appropriato per l&#39;analisi di imaging. Nel nostro laboratorio usiamo Immagine J (Wayne Rosband, National Institutes of Health, Rockville, MD) e personalizzato il software scritto in IDL (Systems Research, Boulder, CO) per quantificare la fluorescenza puncta e le modifiche con Time3. </li></ol> Risultati Represenative La figura 1 mostra FFN511 caricamento in fetta striatale è ora completa. Immagini rappresentative con obiettivo 10x e 60x sono mostrati in Fig.1. L&#39;etichettatura selettiva dei terminali della dopamina può essere decolorato del 20 mM anfetamine. La Figura 2 mostra decolorazione di FFN511 etichettatura KCl alto. La figura 3 mostra decolorazione di FFN511 etichettatura locali stimolazione elettrica. <img src="/files/ftp_upload/1562/1562fig1.jpg" alt="Figura 1" /> Figura 1 FFN511 etichette terminali della dopamina nel vivo fette acuta cortico-striatali (A) Etichettatura di FFN511 in acuto vivere cortico-striatale fetta:.. Etichettatura abbondante nello striato (STR), etichettatura rado nella corteccia (CTX), e nessuna etichetta in corpo calloso (CC). Scala grafica:. 100 micron (B) decolorante della FFN511 dallo striato da anfetamine. Pannelli di sinistra: prima di anfetamine; pannelli di destra: dopo 20 minuti di 20 mM anfetamine. Scala grafica: 10 micron. <img src="/files/ftp_upload/1562/1562fig2.jpg" alt="Figura 2" /> Figura 2. Decolorante di FFN511 etichettatura KCl alto. Etichettatura FFN511 è decolorato entro 2 minuti di applicazione mM KCl 70 in ACSF. Scala grafica: 10μm. <img src="/files/ftp_upload/1562/1562fig3.jpg" alt="Figura 3" /> Figura 3. Dipendente dalla frequenza di decolorazione FFN511 etichettatura striato. (A) stimolazione locale a 4 Hz portato decolorazione dai terminali. Stimolazione iniziata al tempo t = 0. Scala grafica: 5 micron (B) decolorante di FFN511 a 4 Hz è Ca 2 + - e dipendente dalla frequenza.. Controlli ricevuto alcuna stimolazione (153 puncta da 3 fette). Decolorazione con cloruro di cadmio (200 mM) era identico a controlli non stimolate (475 puncta da 5 fette). Le curve di decolorazione per ogni frequenza di stimolazione erano idonei da una singola funzione di decadimento esponenziale e l&#39;emivita (t 1 / 2) valori calcolati come τ x 0,693 (1 Hz: 765 puncta da 9 fette, 4 Hz: 410 puncta da 7 fette, 20 Hz: 416 puncta da 6 fette). Si prega di fare riferimento al microscopio video da 2-fotone microscopia di FFN511 decolorazione durante esocitosi in un preparato striato fetta cervello.

<ol>
	<li>Franklin, K. B. J., Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The mouse brain in stereotaxi coordinators. , (1997).</li><li>Kay, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+synaptic+activity+in+intact+brain+and+slices+with+FM1-43+in+C.+elegans,+lamprey,+and+rat.">Imaging synaptic activity in intact brain and slices with FM1-43 in C. elegans, lamprey, and rat.</a> Neuron. 24, 8098-8117 (1999).</li><li>Bamford, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterosynaptic+dopamine+neurotransmission+selects+sets+of+corticostriatal+terminals.">Heterosynaptic dopamine neurotransmission selects sets of corticostriatal terminals.</a> Neuron. 42, 653-653 (2004).</li></ol>

The authors have nothing to disclose.

In questo video, dimostriamo un metodo per visualizzare neurotrasmissione otticamente con analoghi della dopamina fluorescenti. FFN511 è la prima generazione di FFNs abbiamo sviluppato. Anche se è stato progettato prendendo di mira i neuroni trasportatore vescicolare delle monoamine (VMAT2) che porta neurotrasmettitori monoamine dal citoplasma in vescicole sinaptiche, e le etichette in particolare i terminali della dopamina nello striato e catecolamine presunta e / o terminali della serotonina nella corteccia (come mostrato nella Scienza carta), un periodo di carico appropriata è fondamentale per la specificità in quanto è relativamente FFN511 idrofobo. Abbiamo scoperto che l&#39;incubazione per più di 40 minuti si tradurrà in ampi colorazione aspecifica a fette dello striato. La specificità può essere direttamente determinato da decolorazione con un&#39;alta concentrazione di KCl, che dovrebbe causare il rilascio di FFN all&#39;interno funzionale vescicole sinaptiche. L&#39;esposizione della fetta di FFN511 per meno di 15 minuti si tradurrà in debole segnale fluorescente a causa del carico insufficiente del colorante nei terminali. In questo protocollo, utilizziamo 100μM ADVASEP-7 per rimuovere il colorante legato al tessuto extracellulare. Questo passaggio non è necessario, ma se è omesso, il tempo di washout in ACSF deve essere prolungata. In sintesi, a seconda della preparazione si è scelto, sarà necessario modificare il periodo di concentrazione e di carico per determinare l&#39;etichettatura ottimale.

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>FFN511 (8-(2-Amino-ethyl)-2,3,5,6-tetrahydro-1H,4H-11-oxa-3a-aza-benzo[de]anthracen-10-one)</td>
<td>&nbsp;</td>
<td>Dalibor Sames&#x2019;s laboratory at Columbia University</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>ADVASEP-7 </td>
<td>&nbsp;</td>
<td>CyDex, Overland Park, KS</td>
<td>AR-OA7-005</td>
<td>&nbsp;</td>
<tr>
<td>RC-27L Recording chamber</td>
<td>&nbsp;</td>
<td>Warner Instrument</td>
<td>64-0375</td>
<td>&nbsp;</td>
<tr>
<td>PELCO PrepEze 6-Well Holder</td>
<td>&nbsp;</td>
<td>Ted Pella, Inc.</td>
<td>36157-1</td>
<td>For slice incubation</td>
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<td>Master-8 pulse stimulator and Iso- Flex stimulus isolator</td>
<td>&nbsp;</td>
<td>AMPI, Jerusalem, Israel</td>
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dopamine release at individual presynaptic terminals visualized with ffns

Il sistema nervoso trasmette i segnali tra i neuroni attraverso il rilascio dei neurotrasmettitori durante la fusione delle vescicole sinaptiche. Per osservare l&#39;assorbimento e il rilascio dei neurotrasmettitori dai singoli terminali presinaptici direttamente, abbiamo progettato fluorescenti falsi neurotrasmettitori come substrato per il trasportatore della vescicole sinaptiche monoamine. L&#39;utilizzo di questi sonde per il rilascio di dopamina nello striato immagine, abbiamo fatto alcune osservazioni pertinenti alla plasticità sinaptica. Abbiamo trovato che la frazione delle vescicole sinaptiche per il rilascio dei neurotrasmettitori stimolo era dipendente dalla frequenza dello stimolo. Un cineticamente distinte popolazione &quot;riserva&quot; delle vescicole sinaptiche non è stata osservata in queste condizioni sperimentali. Una frequenza dipendente dalla eterogeneità dei terminali presinaptici stato rivelato che era dipendente in parte sui recettori della dopamina D2, che indica un meccanismo per dipendente dalla frequenza di codifica di selezione presinaptico. Hui Zhang e Niko gubernator G. contribuito in maniera uguale a questo lavoro.

Watch this Scientific Journal Video about Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs at JoVE.com

Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

Un nuovo mezzo per misurare la neurotrasmissione otticamente con analoghi della dopamina fluorescenti.

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 ...

dopamine-release-at-individual-presynaptic-terminals-visualized-with

Research

JoVE Journal

Biology

24.7K Views.  Columbia University. Un nuovo mezzo per misurare la neurotrasmissione otticamente con analoghi della dopamina fluorescenti.

Video: Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

Caricamento terminazioni nervose Drosophila con gli indicatori di calcio

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

Ricerca

Biologia

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

Etichettatura hESC e hMSCs con nanoparticelle di ossido di ferro per i non-invasiva in vivo inseguimento con la RM

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Etichettatura Cellule staminali con le tinture fluorescenti per la rilevazione non invasiva con Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

Primaria dissociato mesencefalo colture cellulari dopamina da roditori neonati

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key symptoms, such as tremor, rigidity and bradikinesia, develop when about 3/4 of dopaminergic cells are lost in the substantia nigra, and fail to provide for the smooth, coordinated regulation of striatal motor circuits. Depression and hallucinations are common, and dementia eventually occurs in 20% of patients. At this time, there is no treatment to delay or stop the progression of PD. Rather, the medications currently available aim more towards the alleviation of these symptoms. New surgical strategies may reversibly switch on the functionally damaged circuits through the electrical stimulation of deep brain structures, but although deep brain stimulation is a major advance, it is not suitable for all patients. It remains therefore necessary to test new cell therapy approaches in preclinical models.
Selective neurotoxic disruption of dopaminergic pathways can be reproduced by injection of 6-hydroxydopamine (6-OHDA) or MPTP (1-methyl-4-phenyl-1,2,3,6-tertahydropyridine) whereas depleting drugs and oxidative-damaging chemicals may also reproduce specific features of PD in rodents. Unlike MPTP, 6-OHDA lesions cause massive irreversible neuronal loss, and can be uni- or bilateral. The 6-OHDA lesion model is reliable, leads to robust motor deficits, and is the most widely used after 40 years of research in rats1. As interactions between grafted cells and host can now be studied more thoroughly in mice rather than in rats, the model has been transposed to mice2,3, where it has been recently characterized4.
In this video, we demonstrate how to lesion the left nigro-striatal pathway of anesthetized mice by slowly delivering 2.0 &mu;L of 6-OHDA through a stereotaxically inserted micro-syringe needle. The loss of dopaminergic input occurs within days, and the functional impairments can be monitored over post-operative weeks and months by rating animal rotations induced by dopaminergic agents5. Here, we show full-body contralateral rotations occurring 10 minutes after a single subcutaneous administration of apomorphine, measured one month after the lesion. Outcomes and drawbacks are discussed below.

Murine Model for Parkinson&#039;s Disease: from 6-OH Dopamine Lesion to Behavioral Test

Parkinson disease is caused by loss of dopaminergic innervation to the striatum, which can be experimentally induced by 6-OH-dopamine. We describe how to perform a stereotaxic lesion and to monitor apomorphine-induced rotational behavior in mice. This model is useful and reliable for testing new therapies for Parkinson disease.

Modello murino per la malattia di Parkinson: da 6-OH lesione dopamina a test comportamentali

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key ...

Cell Electrofusion Visualized with Fluorescence Microscopy

Cell electrofusion is a safe, non-viral and non-chemical method that can be used for preparing hybrid cells for human therapy. Electrofusion involves application of short high-voltage electric pulses to cells that are in close contact. Application of short, high-voltage electric pulses causes destabilization of cell plasma membranes. Destabilized membranes are more permeable for different molecules and also prone to fusion with any neighboring destabilized membranes. Electrofusion is thus a convenient method to achieve a non-specific fusion of very different cells in vitro. In order to obtain fusion, cell membranes, destabilized by electric field, must be in a close contact to allow merging of their lipid bilayers and consequently their cytoplasm. In this video, we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method. In this method, cells are allowed to attach only slightly to the surface of the well, so that medium can be exchanged and cells still preserve their spherical shape. Fusion visualization is assessed by pre-labeling of the cytoplasm of cells with different fluorescent cell tracker dyes; half of the cells are labeled with orange CMRA and the other half with green CMFDA. Fusion yield is determined as the number of dually fluorescent cells divided with the number of all cells multiplied by two.

In this video we demonstrate efficient electrofusion of cells in vitro by means of modified adherence method using electroporation and the subsequent detection of fused cells visualization with fluorescence microscopy.

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 ...

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional regulation1. Therefore, an essential step towards understanding transcript regulation at the molecular level is to gain positional information on the binding sites of RBPs2.
Protein-RNA interactions can be studied using biochemical methods, but these approaches do not address RNA binding in its native cellular context. Initial attempts to study protein-RNA complexes in their cellular environment employed affinity purification or immunoprecipitation combined with differential display or microarray analysis (RIP-CHIP)3-5. These approaches were prone to identifying indirect or non-physiological interactions6. In order to increase the specificity and positional resolution, a strategy referred to as CLIP (UV cross-linking and immunoprecipitation) was introduced7,8. CLIP combines UV cross-linking of proteins and RNA molecules with rigorous purification schemes including denaturing polyacrylamide gel electrophoresis. In combination with high-throughput sequencing technologies, CLIP has proven as a powerful tool to study protein-RNA interactions on a genome-wide scale (referred to as HITS-CLIP or CLIP-seq)9,10. Recently, PAR-CLIP was introduced that uses photoreactive ribonucleoside analogs for cross-linking11,12.
Despite the high specificity of the obtained data, CLIP experiments often generate cDNA libraries of limited sequence complexity. This is partly due to the restricted amount of co-purified RNA and the two inefficient RNA ligation reactions required for library preparation. In addition, primer extension assays indicated that many cDNAs truncate prematurely at the crosslinked nucleotide13. Such truncated cDNAs are lost during the standard CLIP library preparation protocol. We recently developed iCLIP (individual-nucleotide resolution CLIP), which captures the truncated cDNAs by replacing one of the inefficient intermolecular RNA ligation steps with a more efficient intramolecular cDNA circularization (Figure 1)14. Importantly, sequencing the truncated cDNAs provides insights into the position of the cross-link site at nucleotide resolution. We successfully applied iCLIP to study hnRNP C particle organization on a genome-wide scale and assess its role in splicing regulation14.

The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.

iClip - trascrittoma livello Mappatura delle interazioni proteina-RNA con risoluzione individuale Nucleotide

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional ...

Preparing Individual Drosophila Egg Chambers for Live Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, cell differentiation and organogenesis 1. Correct preparation of the experimental samples is a crucial, often neglected, step. The goal of preparation is to ensure physiological relevance and to establish optimal imaging conditions. To maintain tissue viability, it is critical to avoid dehydration, hypoxia, overheating or medium deterioration 2. 
 The Drosophila egg chamber is a well established system for examining questions relating, but not limited, to body patterning, mRNA localization and cytoskeletal organization 3,4. For early- and mid-stage egg chambers, mounting in halocarbon oil is good for survival in that it allows free diffusion of oxygen, prevents dehydration and hypoxia and has superb optical properties for microscopy. Imaging of fluorescent proteins is possible through the introduction of transgenes into the egg chamber or physical injection of labeled RNA, protein or antibodies 5-7. For example, addition of MS2 constructs to the genome of animals enables real time observation of mRNAs in the oocyte 8. These constructs allow for in vivo labeling of mRNA through utilization of the MS2 bacteriophage RNA stem loop interaction with its coat protein 9. 
 Here, we present a protocol for the extraction of ovaries as well as isolating individual ovarioles and egg chambers from the female Drosophila. For a detailed description of Drosophila oogenesis see Allan C. Spradling (1993, reprinted 2009) 10.

Preparing Individual Drosophila Egg Chambers for Live Imaging

The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.

Preparazione individuale Drosophila Egg Chambers per l&#39;imaging dal vivo

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

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 ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

Imaging di fluorescenza con One nanometri Precisione (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...