D. Şemeş G. Harold &amp; Leila Y. Vakfı ve Columbia Üniversitesi Fen ve Mühendislik Girişimleri Mathers teşekkürler. D. Şemeş ve D. Sulzer Nörobilim Ödülü McKnight Teknolojik Yenilikler McKnight Vakfı teşekkür D. Sulzer NIDA, NIMH ve Picower ve Parkinson Hastalığı Vakıflar teşekkürler. H. Zhang sayesinde NARSAD. RH Edwards, Michael J. Fox Vakfı, Ulusal Parkinson Vakfı, NIDA ve NIMH teşekkürler. 6-OHDA enjeksiyonları ve tavsiyeleri, yararlı tartışma, görüntüleme analiz programlama için Merek Siu Mark Sonders ve Jan Schmoranzer Robert Burke TIRF mikroskopi kurulum, teknik destek için teşekkür ederiz.

Bu yöntem bildirilen araştırma <a href="http://www.sciencemag.org/cgi/content/full/324/5933/1441">Gubernator ve ark, Bilim 324 (5933). 1441-1444 (2009)</a> . 1. Akut striatal dilim hazırlanması <ol><li> Akut striatal dilim hazırlamadan önce, bir oksijen yapay beyin omurilik sıvısı (ASCF) ve önce beyin çıkarımı için en az 15 dakika boyunca buz ile soğuk olmalıdır. ACSF buz tutun ve diseksiyon sırasında oksijenli. ACSF (mM): NaCl 125, 2.5 KCl, NaHCO 3 26, CaCl 2 2.4 MgSO4 1.3, KH 2 PO 4 0.3, glukoz 10, Hepes 5; pH 7,3-7,4 290-295 mOsm. </li><li> Anestezi olmadan erkek fare başını kesmek. </li><li> Fare tüm beyin ayıklayın ve Krazy ® Yapıştırıcı kullanarak vibratome yerleştirilen vibratome tepsi üzerine monte. Beyin çıkarma ve montajı, 2 dakika içinde yapılmalıdır. Bu süre zarfında, ACSF buz tutmak ve doku sağlıklı tutmak için oksijenli emin olun. </li><li> Bregma 1,54 arasında 0,62 1 250μm kalınlığında koronal striatal beyin dilimleri kesin. Üç bütün dilimleri daha sonra bir iğne ile 6 yarım dilimler halinde kesilmiş olabilir elde edilecektir. </li><li> Prob yüklemeden önce en az 1 saat oda sıcaklığında oksijenli ACSF beyin dilimleri inkübe edin. Dilimler iyi FFN511 yükleme ekranı ve 4 saat sonra görüntüleme dilim hazırlanması için aktif kalır. </li></ol> 2. FFN511 Yükleniyor <ol><li> FFN511 yükleme solüsyonu (içinde 10μM FFN511 ACSF) hazırlayın; ACSF içinde ADVASEP-7 100μM hazırlamak. Tüm çözümler taze olarak hazırlanır ve en az 15 dakikalık dilimler halinde yüklemeden önce oksijenli olmalıdır. </li><li> Bireysel dilim FFN511 yükleme çözümü ile oda sıcaklığında 30 dakika inkübe edin. </li><li> Sonra oksijenli 100μM ADVASEP-7 ACSF yüklenen dilim az 30 dakika oda sıcaklığında 2 kuluçka ekstraselüler dokuya bağlı boya çıkarın. Dilimler görüntüleme için hazırız. </li></ol> 3. Görüntüleme FFN511 beyin dilimleri Dilimleri görüntüleme FFN511 multiphoton lazer tarama mikroskobu kullanılarak yapılır. Biz Prairie Ultima multiphoton lazer tarama mikroskop kullanılarak (Biz daha önce bir Zeiss LSM 510 NLO multiphoton lazer tarama mikroskobu kullanılarak ve Zeiss mikroskop ile bazı sonuçlar elde edilmiştir). <ol><li> Kayıt odasında FFN511 yüklü dilim (RC-27L, Warner) yerleştirin ve bir akış hızı dakikada 1-2 ml az oksijenli ACSF superfuse. Sonra deney sırasında hareketini en aza indirmek için, dilimin üstüne naylon dizeleri ile platin arp yerleştirin. Hareketi daha da en aza indirmek için, dilim odasında en az 10 dakika önce görüntüleme stabilize olması için izin verir. </li><li> Görselleştirin ve 10x suya daldırma hedefi altında parlak bir alan lüminesans dorsal striatum bulmak. </li><li> Yeri ve konumu bu bölgede bipolar bükülmüş tungsten elektrot stimülasyon deneyi gerçekleştirmek durumunda. Iki uyarıcı bipolar elektrot uçları arasındaki 300 mikron ideal görüntüleme alanının içinde yer almaktadır. Görme alanının merkezinde bu bölgeye yerleştirin. </li><li> Resim FFN511 bir 63x (0.9 NA) su altında kalacak ultraviyole amacı altında striatal terminalleri etiketli. FFN511 Spektral Fizik ve striatal terminalleri optimal floresan Mai Tai lazer 760 nm dalga boyunda bir bant geçiren filtre (480-520 nm) ile görülür heyecanlı. Görüntüler, 12-bit formatında 512 x 512 piksel çözünürlük faiz 75 x 75 mikron bölgeleri ile yakalanır. Uyarılmış destaining deneyler için, z-düzleminde 1 mikron ayrılmış z-eksen kayması, 5-7 görüntüleri z serisi, telafi etmek için her dönem için elde edilir. </li></ol> 4. FFN511 Destaining FFN511 etiket KCl (70 mM KCl) yüksek konsantrasyon (+) amfetamin sülfat (AMPH, 20 mcM), ya da elektrik stimülasyonu ya destained olabilir. Yüksek potasyum ACSF çözüm beyin dilim uygulanır KCl destaining deney, 2 dakika içinde FFN511 etiket destains. Tutanak xyz-t görüntüleri zaman içinde destaining FFN511 izlemek için. Aşağıdaki elektrik stimülasyonu-bağımlı dopamin terminali destaining açıklar: <ol><li> Zamanla FFN511 destaining izlemek için, xyz-t görüntüleri kaydedildi. Dilim (z-ekseninde az 4 mm) en az hareket sağlamak için ve hiçbir z yığının lazer, kontrol zaman serisi görüntüleri photobleaching stimülasyon öncesinde en az 5 dakika süreyle alınmalıdır. Aksi takdirde, lazer gücü ayarlayın. </li><li> 1 frekansları, 4 ya da 20 Hz uyarılmaya başlanması ise bu kontrol görüntülerinin ardından, xyz-t görüntüleme devam etmektedir. 1, 4 veya 20 Hz (300 μs x 1 mA) Uyaranlar bipolar elektrotlar kullanarak Master-8 darbe jeneratörü (Ampi, Kudüs, İsrail) tarafından tetiklenen bir Iso-Flex uyaran izolatör yerel striatum uygulanır. En aza indirmek içinserbest sitelerin depolarizasyon varyasyonu, siklik voltametri tarafından belirlenen maksimal stimülasyon yoğunluğu% 150 uygulama stimülasyon protokolü kullanılır. </li><li> Görüntü analiz için uygun yazılımı kullanın. Bizim laboratuvarda, biz IDL Görüntü J (Wayne Rosband, Ulusal Sağlık Enstitüleri, Rockville, MD) ve özel yazılmış yazılımlar (Araştırma Sistemleri, Boulder, CO) Süresi3 puncta floresan ve değişiklikleri ölçmek için kullanın. </li></ol> Represenative Sonuçlar Şekil 1 striatal dilim içine FFN511 yükleme işlemi tamamlandıktan gösterir. 10x ve 60x objektif kullanarak Temsilcisi görüntüleri Şekil 1&#39;de gösterilmiştir. Dopamin terminalleri seçici etiketleme 20 mcM amfetamin destained olabilir. Yüksek KCl FFN511 etiketleme destaining Şekil 2 göstermektedir. Şekil yerel elektrik stimülasyonu ile etiketleme FFN511 destaining 3 gösterir. <img src="/files/ftp_upload/1562/1562fig1.jpg" alt="Şekil 1" /> Şekil 1 canlı kortikal-striatal akut dilim FFN511 etiketler dopamin terminalleri (A) akut kortikal-striatal canlı dilim FFN511 Etiketleme:. Striatum bol etiketleme (STR), korteks seyreldiğinde etiketleme (CTX) ve herhangi bir etiket korpus kallozum (CC). Ölçek çubuğu: 100 mm (B) Destaining amfetamin tarafından striatum FFN511 Sol paneller: amfetamin önce sağ panelleri: 20 mcM amfetamin 20 dakika sonra. Ölçek çubuğu: 10 mm. <img src="/files/ftp_upload/1562/1562fig2.jpg" alt="Şekil 2" /> Şekil 2 yüksek KCl FFN511 etiketleme Destaining. FFN511 etiketleme ACSF 2 dk 70 mM KCl uygulama içinde destained. Ölçek çubuğu: 10μm. <img src="/files/ftp_upload/1562/1562fig3.jpg" alt="Şekil 3" /> Şekil 3 striatumda FFN511 etiketleme Frekans bağımlı destaining (A) 4 Hz Yerel uyarılması terminallerinden destaining sonuçlandı. Uyarım t = 0 &#39;da başladı. Ölçek çubuğu: 5 mikron (B) 4 Hz az FFN511 Destaining Ca 2 + ve frekans bağımlı. Kontroller hiçbir stimülasyonu (3 dilim 153 puncta) aldı. Kadmiyum klorür (200 mcM) ile Destaining unstimulated denetimleri (5 dilim 475 puncta) aynıydı. Olarak hesaplanan değerler her bir uyarımı frekans için destaining eğrileri tek bir üstel bozulma fonksiyonu ve yarılanma ömrü (t 1 / 2) uygun τ x 0.693 (1 Hz: 9 dilimleri, 4 Hz 765 puncta: 7 dilim 410 puncta 20 Hz: 6 dilim) 416 puncta. Striatal beyin dilim hazırlık ekzositoz sırasında FFN511 destaining 2-foton mikroskopi video mikrografı bakın.

<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.

Bu video, floresan dopamin analogları kullanarak optik nörotransmisyon görselleştirmek için bir yöntem ortaya koymaktadır. FFN511 FFNs ilk nesil biz geliştirdik. Bilim gösterildiği gibi sinaptik veziküller içine sitoplazma monoamin nörotransmitterler taşır ve özellikle korteks, striatum ve varsayılan katekolamin ve / veya serotonin terminal dopamin terminalleri etiketler nöronal veziküler monoamin taşıyıcı (VMAT2) (hedefleyerek tasarlanmış olmasına rağmen FFN511 nispeten hidrofobik olduğundan kağıt), uygun bir yükleme süresi özgüllük için kritik öneme sahiptir. Biz 40 dakika daha uzun kuluçka striatal dilimleri geniş nonspesifik boyanma neden olacaktır bulundu. Özgüllüğü ve sade bir dille, fonksiyonel sinaptik veziküller içinde FFN serbest bırakılması neden KCl yüksek bir konsantrasyon ile destaining tespit edilebilir. Az 15 dakika süreyle FFN511 dilim Pozlama terminallerde boya yetersiz yükleme nedeniyle zayıf floresan sinyal neden olur. Bu protokol, ekstrasellüler doku bağlı boya kaldırmak için 100μM ADVASEP-7. Bu adım gerekli değildir, ancak belirtilmezse, ACSF içinde arınma süresi uzamış olması gerekir. Özetle, seçtiğiniz hazırlanmasına bağlı olarak, en iyi etiketleme belirlemek için konsantrasyon ve yükleme süresini değiştirmek için ihtiyacınız olacak.

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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>
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<td>ADVASEP-7 </td>
<td>&nbsp;</td>
<td>CyDex, Overland Park, KS</td>
<td>AR-OA7-005</td>
<td>&nbsp;</td>
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<td>RC-27L Recording chamber</td>
<td>&nbsp;</td>
<td>Warner Instrument</td>
<td>64-0375</td>
<td>&nbsp;</td>
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<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

Sinir sistemi nöronlar arasındaki sinaptik vezikül füzyon sırasında nörotransmiter bülteni ile sinyalleri iletir. Nörotransmitter alımı ve doğrudan bireysel presinaptik terminallerden serbest gözlemlemek için, floresan yanlış nörotransmitterlerin sinaptik vezikül monoamin taşıyıcı yüzeylerde olarak tasarlanmıştır. Striatum görüntü dopamin serbest bırakmak için bu problar kullanarak, sinaptik plastisite ile ilgili çeşitli gözlemler yapmıştır. Biz uyaran ortalama serbest nörotransmitter sinaptik veziküllerin kesir uyaran frekansı bağımlı olduğu bulundu. Bu deneysel koşullar altında kinetik farklı bir &quot;rezerv&quot; sinaptik vezikül nüfus gözlenmemiştir. Presinaptik terminallerin bir frekans bağımlı heterojenite presinaptik seçim frekans bağımlı kodlama için bir mekanizma gösteren, D2 dopamin reseptörlerine parçası bağımlı ortaya çıktı. Hui Zhang ve Niko G. Gubernator bu iş için eşit olarak katkıda bulundu.

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

Yeni bir floresan dopamin analogları kullanarak optik nörotransmisyon ölçmek anlamına gelir.

Bireysel Presinaptik Terminalleri Dopamin Yayın FFNs ile görüntülendi

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 Görüntüleme.  Columbia University. Yeni bir floresan dopamin analogları kullanarak optik nörotransmisyon ölçmek anlamına gelir.

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.

Kalsiyum Göstergeleri ile yükleme Drosophila Sinir Terminalleri

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 ...

Biyoloji

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.

Demir Oksit hESC ve hMSCs Etiketleme MR Görüntüleme in vivo Takip Non-Invasive Nanopartiküller

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.

Optik Görüntüleme ile non-invaziv Algılama için Floresan Boyalar Kök Hücreler Etiketleme

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.

Kemirgen Yenidoğanlar İlköğretim grubuyla orta beyin dopamin Hücre Kültürleri

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.

Parkinson Hastalığı için murin Model: 6-OH Dopamin Lezyon Davranış Testi

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.

Hücre Elektrofüzyon Floresan Mikroskop ile görüntülendi

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 - Bireysel Nükleotid Çözünürlük protein-RNA Etkileşimleri Transcriptome geniş Haritalama

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.

Bireysel hazırlanması Drosophila</emCanlı Görüntüleme için> Yumurta Chambers

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.

Tütün protoplastlarında ve Yapraklar Bimoleküler Floresan tamamlama tarafından Visualized protein-protein etkileşimleri

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.

Bir nanometre Hassasiyet Floresans Görüntüleme (Fiona)

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