Tüm St Louis Kardiyak Moleküler Mühendislik Lab Washington Üniversitesi&#39;nden üyeleri. Bir Burroughs Bilimsel Arakesitte Fonu Kariyer Ödülü Hoşgeldiniz - 1010299 (JS).

<ol>
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U.S.A. 110, 8272-8277 (2013).</li><li>Stefani, E., Bezanilla, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cut-open+oocyte+voltage-clamp+technique.">Cut-open oocyte voltage-clamp technique.</a> Methods Enzymol. 293, 300-318 (1998).</li><li>Muroi, Y., Chanda, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+anesthetics+disrupt+energetic+coupling+between+the+voltage-sensing+segments+of+a+sodium+channel.">Local anesthetics disrupt energetic coupling between the voltage-sensing segments of a sodium channel.</a> J. Gen. 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Yazarlar ifşa hiçbir şey yok.

Kesim açık oosit Vazelin boşluk gerilim kelepçe tekniği hızlı veri çözünürlüğü sağlar, düşük gürültü, nispeten uzun protokolleri üzerinden 19 iç çözümü ve dış çözüm kompozisyon üzerinde kontrol ve istikrarlı kayıtları arttı. Bu avantajlar, ayrı Standart iki elektrotlu voltaj kelepçe ve yama kelepçe teknikleri bu teknik ayarlayın. Özel ekipman gerekli ve protokol nispeten zor olmasına rağmen, sistem optimize edildikten sonra, çok az sayıda sorunlar oluşur. Bu sody...

Uzman voltaj kenetleme teknikleri, kontrol edilen membran potansiyelleri iyonik akımların kayıt izin verir. Yaygın iki elektrotlu voltaj kelepçe (TEVC) kullanılmış ve yama kelepçe teknikleri çok iyon kanallarının özellikleri hakkında güvenilir elektrofizyolojik bilgi sağlar. Ancak, bu yöntemlerin her ikisi de hızlı voltaj-kapılı sodyum kanalları ve bu tür Xenopus oosit gibi zarlarında diğer hızlı aktive kanallar için güvenilir verilerin ele geçirilmesini önlemek dezavantajları var. Bezanilla ve Stefani laboratuvarları dolayısıyla oosit 2 için kesim açık Vazelin boşluk gerilim kelepçe tekniği (COVG) geliştirdi. Bu teknik, Na +, K + ve Ca 2 + channels 3-8 kaydetmek için yaygın olarak uygulanmaktadır. COVG kayıt sırasında, bir heterolog protein ifade oosit zarı üç bölgeye ayrılmıştır. Iyonik mevcut veriler olarak oositin üst bölgesinden kaydedilirüst bölgeyi çevreleyen banyo kolayca ve hızlı bir şekilde değiştirilebilir bir komut potansiyeli, kenetlenir. Orta bölge üst bölge 9 ile aynı potansiyele kenetlenir edilerek kaçak akımlara karşı koruma sağlar. Oosit açıklık (cut-açık) bir saponin çözeltisi ya da bir kanül aracılığıyla oluştuğu alt bölgesidir. Kimyasal ya da alt bölgesinde zarın manuel açma toprağa kenetlenir iç potansiyel kontrolünü sağlar ve alt bölme çözeltisi ile hücre iç sürekliliğini hale getirir. En iyi odasındaki çözelti değişimi dış çevresi değiştirir, oysa alt odasına çözümler perfüzyon, iç ortam özelliklerini ayarlayabilirsiniz. <img alt="Şekil 1" fo:content-width="5in" fo:src="/files/ftp_upload/51040/51040fig1highres.jpg" src="/files/ftp_upload/51040/51040fig1.jpg" width="500px" /> Şekil 1. Oosit Cut-Açık Gerilim-Kelepçe Banyo Kurulum Şeması. (A) Üstbirbirinden ayrılmış üç banyolarının görünümünde aşağı. COVG için bölmelerin boyutları şekil üzerinde görüntülenir. (B) testi pozisyonda hamam kurulum yan görünümü. <a href="http://www.jove.com/files/ftp_upload/51040/51040fig1highres.jpg" target="_blank">resmi büyütmek için buraya tıklayın</a> . COVG tekniğin avantajları, düşük akım gürültü (3 kHz 1 nA), dış ortamın iyonik kompozisyonu, iç ortam, hızlı zamanı çözünürlük (çürüme 20-100 mikro saniye olduğu zaman sabiti modüle yeteneği kontrolü, kapasite geçici), ve birkaç saat 9 stabil kayıtları. Dezavantajları özel ekipman gerektirir ve iki elektrotlu voltaj kelepçeleme (TEVC) 10 ile karşılaştırıldığında gerçekleştirmek için daha zor olmasıdır. COVG yaklaşımı son derece özel ekipman ve karmaşık usul unsurları gerektirir iken, valu edinimi için izin verebilirmümkün elektrofizyolojik veriler. Böyle hızlı kinetik ve kuyruk akımları 4 ile akımlarını yolluk gibi bu veriler, kanal köhne dahil olmak üzere diğer voltaj sıkıştırma protokolleri ile ilgili bazı sorunları olmadan kaydedilebilir. COVG kurulum için küçük değişiklikler sıcaklık kontrolörleri ve gerilim kelepçe fluorometri (VKF) kullanımı için izin verebilirsiniz. COVG montaj içinde gerilim kelepçe fluorometri unsurların eklenmesi anda mevcut 11-13 kaydederken protein yapısal değişimleri izlemek için yeteneğini kazandıran veri çıkışını güçlendirir.

<table border="0" cellpadding="0" cellspacing="0" width="515">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">External Solution</td>
			<td style="width:131px;">Brand</td>
			<td style="width:72px;">Catalog Number</td>
			<td style="width:124px;">[Final], weight, or volume</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">N-methyl-D-glucamine (NMDG)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">M2004</td>
			<td style="width:124px;">25mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">MES Sodium Salt</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">M5057</td>
			<td style="width:124px;">90mM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">HEPES</td>
			<td style="width:131px;">Research Products International</td>
			<td style="width:72px;">H75030</td>
			<td style="width:124px;">20mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Calcium hydroxide</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">239232</td>
			<td style="width:124px;">2mM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">MES Hydrate</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">M8250</td>
			<td style="width:124px;">variable (pH to 7.4)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;"></td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Internal Solution</td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">N-methyl-D-glucamine (NMDG)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">M2004</td>
			<td style="width:124px;">105mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">MES Sodium Salt</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">M5057</td>
			<td style="width:124px;">10mM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">HEPES</td>
			<td style="width:131px;">Research Products International</td>
			<td style="width:72px;">H75030</td>
			<td style="width:124px;">20mM</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:189px;">Ethylene glycol-bis(2-aminoethylether)-N,N,N&#39;,N&#39;-tetraacetic acid (EGTA)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">E4378</td>
			<td style="width:124px;">2mM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">MES Hydrate</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">M8250</td>
			<td style="width:124px;">variable (pH to 7.4)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;"></td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Depolarizing Solution</td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">KCl</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">221473</td>
			<td style="width:124px;">110mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Magnesium chloride</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">M8266</td>
			<td style="width:124px;">1.5mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Calcium Chloride</td>
			<td style="width:131px;">Caisson</td>
			<td style="width:72px;">C021</td>
			<td style="width:124px;">0.8mM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">HEPES</td>
			<td style="width:131px;">Research Products International</td>
			<td style="width:72px;">H75030</td>
			<td style="width:124px;">10mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;"></td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Pipet Solution</td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">KCl</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">221473</td>
			<td style="width:124px;">3M</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:189px;"></td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:189px;">Saponin Solution</td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Saponin</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">47036</td>
			<td style="width:124px;">0.125g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Internal Solution</td>
			<td style="width:131px;">See above</td>
			<td style="width:72px;"></td>
			<td style="width:124px;">50mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;"></td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;"></td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;"></td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Agar Bridge Solution</td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">N-methyl-D-glucamine (NMDG)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">M2004</td>
			<td style="width:124px;">100ml of 1M</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">HEPES</td>
			<td style="width:131px;">Research Products International</td>
			<td style="width:72px;">H75030</td>
			<td style="width:124px;">1.2g</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">MES Hydrate</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">M8250</td>
			<td style="width:124px;">variable (pH to 7.4)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">Granulated Agar</td>
			<td style="width:131px;">Research Products International</td>
			<td style="width:72px;">A20250</td>
			<td align="right" style="width:124px;">3%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;"></td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">NMDG Storage Solution</td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">NMDG, HEPES, MES Hydrate solution</td>
			<td style="width:131px;">see above</td>
			<td style="width:72px;"></td>
			<td style="width:124px;">40ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Water</td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;">60ml</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">Name of Material/ Equipment</td>
			<td style="width:148px;">Company</td>
			<td style="width:144px;">Catalog Number</td>
			<td style="width:285px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">High Performance Oocyte Clamp</td>
			<td style="width:148px;">Dagan</td>
			<td style="width:144px;">CA-1B</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">Data Acquisition System</td>
			<td style="width:148px;">Axon CNS</td>
			<td style="width:144px;">&#160;Digidata 1440A</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">Oscilloscope</td>
			<td style="width:148px;">Tektronix&#160;</td>
			<td style="width:144px;">TDS 210</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">Rack Power Filter</td>
			<td style="width:148px;">APC&#160;</td>
			<td style="width:144px;">G5</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:149px;">Heating/Cooling Bath Temperature Controller</td>
			<td style="width:148px;">Dagan</td>
			<td style="width:144px;">HCC-100A</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">PC</td>
			<td style="width:148px;">Dell</td>
			<td style="width:144px;">Optiplex 990</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">pCLAMP 10.3 Voltage Clamp Software</td>
			<td style="width:148px;">Molecular Devices, LLC</td>
			<td style="width:144px;">pCLAMP10.3</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:149px;">TMC Vibration Control TableTop Platform</td>
			<td style="width:148px;">TMC</td>
			<td style="width:144px;">64 SERIES</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">TMC Vibration Control Air Table</td>
			<td style="width:148px;">TMC</td>
			<td style="width:144px;"><a name="RANGE!C10">20 Series&#160;</a></td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">V1/I Electrode Data Collector</td>
			<td style="width:148px;">Dagan</td>
			<td style="width:144px;">part of CA-1B</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">MX10L Micromanipulator</td>
			<td style="width:148px;">Siskiyou</td>
			<td style="width:144px;">MX10L</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:149px;">Bath/Guard (I/V) Headstage (with appropriate connectors)</td>
			<td style="width:148px;">Dagan</td>
			<td style="width:144px;">part of CA-1B</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">Microscope</td>
			<td style="width:148px;">Omano</td>
			<td style="width:144px;">OM2300S-JW11</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:149px;">Temperature Control Bath</td>
			<td style="width:148px;">Custom or Dagan</td>
			<td style="width:144px;">Custom or HE-204C</td>
			<td style="width:285px;">Custom chamber made from materials from Cool Polymers (D-series). Dagan also provides a prefeabricated stage (HE-204C).</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">Custom AgCl Pellet Container</td>
			<td style="width:148px;">Custom</td>
			<td style="width:144px;">Custom</td>
			<td style="width:285px;">Custom machined</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">Ag/AgCl electrode, pellet, 2.0 mm</td>
			<td style="width:148px;">Warner</td>
			<td style="width:144px;">E-206</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">External Oocyte Bath</td>
			<td style="width:148px;">Custom or Dagan</td>
			<td style="width:144px;">Custom or CC-1-T-LB</td>
			<td style="width:285px;">Custom machined or purchased from Dagan</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">Internal Oocyte Bath</td>
			<td style="width:148px;">Custom or Dagan</td>
			<td style="width:144px;">Custom or CC-TG-ND</td>
			<td style="width:285px;">Custom machined or purchased from Dagan</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:149px;">Capillaries for Agar Bridges and Pulled Electrodes</td>
			<td style="width:148px;">Warner</td>
			<td style="width:144px;">G150T-4</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:149px;">Rotatable Mounts for the Microscope, Micromanipulator, and Bath</td>
			<td style="width:148px;">Siskiyou</td>
			<td style="width:144px;">SD-1280P</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">Fiber-Lite</td>
			<td style="width:148px;">Dolan-Jenner</td>
			<td style="width:144px;">LMI-600</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">Regular Bleach</td>
			<td style="width:148px;">Clorox</td>
			<td style="width:144px;">470174-764</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">Xenopus laevis Oocytes</td>
			<td style="width:148px;">Nasco</td>
			<td style="width:144px;">LM535M (sexually mature females)</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:149px;">90 Na+ External Solution</td>
			<td style="width:148px;">See Solutions sheet</td>
			<td style="width:144px;"></td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:149px;">10 Na+ Internal Solution</td>
			<td style="width:148px;">See Solutions sheet</td>
			<td style="width:144px;"></td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">3 M KCL</td>
			<td style="width:148px;">See Solutions sheet</td>
			<td style="width:144px;"></td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">Saponin</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:144px;">47036</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">NMDG Storage Solution</td>
			<td style="width:148px;">See Solutions sheet</td>
			<td style="width:144px;"></td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">5mL transfer pipets</td>
			<td style="width:148px;">SciMart</td>
			<td style="width:144px;">GS-52</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:149px;">Modified KCl electrode injector</td>
			<td style="width:148px;">BD</td>
			<td style="width:144px;">309659</td>
			<td style="width:285px;">Plastic syringe tip melted to allow for injection of solution into electrodes. Alternatively, a Microfil by WPI can be purchased.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">Microvaccum</td>
			<td style="width:148px;">Custom</td>
			<td style="width:144px;">Custom</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">Forceps</td>
			<td style="width:148px;">VWR</td>
			<td style="width:144px;">63040-458</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">Oocyte Handling Tools (Pipette Pump)</td>
			<td style="width:148px;">VWR</td>
			<td style="width:144px;">53502-222</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">Deionized Water Squirt Bottle</td>
			<td style="width:148px;">VWR</td>
			<td style="width:144px;">16649-911</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">Vaseline Petroleum Jelly</td>
			<td style="width:148px;">Fisher Scientific</td>
			<td style="width:144px;">19-086-291&#160;</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;"></td>
			<td style="width:148px;"></td>
			<td style="width:144px;"></td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:149px;">Additional Materials Required for VCF Recordings:</td>
			<td style="width:148px;"></td>
			<td style="width:144px;"></td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">VCF Microscope</td>
			<td style="width:148px;">Nikon</td>
			<td style="width:144px;">Eclipse FN1</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">Nikon CFI APO 40XW NIR Objective</td>
			<td style="width:148px;">Nikon</td>
			<td style="width:144px;">N40X-NIR</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:149px;">X-Y Translator System for Fixed-Stage Upright Microscopes</td>
			<td style="width:148px;">Sutter Instruments</td>
			<td style="width:144px;">MT500-586</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">External VCF Oocyte Bath</td>
			<td style="width:148px;">Custom</td>
			<td style="width:144px;"></td>
			<td style="width:285px;">Custom machined. The chamber dimensions are 2.7 x 1.9 x 0.4 cm.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">Internal VCF Oocyte Bath</td>
			<td style="width:148px;">Custom</td>
			<td style="width:144px;"></td>
			<td style="width:285px;">Custom machined. The chamber dimensions are 1.6 x 1.6 x 0.4 cm.</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:149px;">Modified Temperature Control Bath</td>
			<td style="width:148px;">Custom</td>
			<td style="width:144px;"></td>
			<td style="width:285px;">Custom chamber made from materials from Cool Polymers (D-series). The chamber dimensions of the modified temperature controller bath are 2.7 x 1.9 x 0.3 cm for the horizontal chamber, and 1 x 2.5 x 0.5 cm for the vertical chamber.</td>
		</tr>
	</tbody>
</table>

the xenopus oocyte cut-open vaseline gap voltage-clamp technique with fluorometry

Kesik açık oosit Vaseline boşluk (COVG) voltaj kelepçe tekniği oositlerde heterolog iyon kanalları, elektrofizyolojik ve kinetik özelliklerinin analizi sağlar. Kesim açık kurulumdan Kayıtlar düşük büyüklüğü yolluk akımları, hızlı iyonik akım aktivasyonu ve deaktivasyonunu çözmek için özellikle yararlıdır. Iki elektrotlu voltaj kelepçe (TEVC) tekniği fazla temel faydaları artış kelepçe hızı, gelişmiş bir sinyal-gürültü oranı ve hücre içi ve hücre dışı ortamı ayarlama yeteneği de bulunmaktadır. Burada, kesme açık setup ve protokol hem de voltaj kelepçe fluorometri özelliği eklemek için gerekli olan değişiklikleri göstermek için, (HNA V 1.5), Xenopus oositlerde eksprese edilen insan kardiyak sodyum kanalı kullanır. Böyle HNA V 1.5 gibi hızlı aktive iyon kanalları, özellikleri, tam olarak hangi de, TEVC kullanılarak oda sıcaklığına yakın çözülemezoosit membran saat tamamı voltaj kontrolü zorlaştırır, kenetlenir. Bununla birlikte, kesme, açık teknik, hücre zarının sadece küçük bir kısmının izolasyon patch clamp teknikleriyle ilişkili kanal halsiz önlerken doğru hızlı kinetiği kaydetmek için gerekli olan hızlı bir sıkma sağlar. COVG teknik, iyon kanalı kinetik ve elektrofizyolojik özellikleri ile bağlantılı olarak daha fazla protein, hücre dışı hareket uygulanır fluoroforlar, genetik olarak kodlanmış floresan proteinleri sokulması, ya da doğal olmayan amino asitlerin dahil sistein konjügasyonu ile izlenen voltaj kelepçe fluorometri kullanılarak deneye tabi tutulabilir ilgi 1&#39;in bölgesine. Bu, ek veri flüoresan molekülü çevreleyen mikro-değişimlere ile proteinin voltaj bağımlı konformasyonel yeniden düzenlemeleri ile ilgili kinetik bilgi verir.

Şekil 4, görüntüler bir saponin çözeltisi olarak oositin geçirgenlikteki değişim oositin alt kısmına uygulanır. Nüfuziyet 5 saponin aşağıdaki difüzyon ile hücre içi çözelti değişim oranını göstermektedir Şekil. 20-40 dk bir kararlı duruma 2,18 gelmek için gereklidir. Kayıt protokol oluşturulan Şekil 6A gösterisi izler. Şekil voltaj protokolü (ek) tepki olarak (P/-8 sızıntı ç?...

Watch this Scientific Journal Video about The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry at JoVE.com

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

Kesik açık vazelin boşluk yaklaşım hızlı kanal kinetiği yüksek çözünürlüklü Xenopus oositlerde eksprese edilen voltaj-bağımlı iyon kanallarının iyonik ve yolluk akımlarının düşük ses kayıtları elde etmek için kullanılır. Küçük değişiklikler ile, voltaj kıskaç fluorometri kesme açık oosit protokolüne akuple edilebilir.

<em&gt; Xenopus</emFluorometri ile&gt; Yumurta Kes-açık Vazelin Gap Gerilim-kelepçe Tekniği

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

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Unit 1

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

Root-Locus Method

SCIENTISTS IN ACTION

18.3K Görüntüleme.  Washington University in St. Louis.  Kesik açık vazelin boşluk yaklaşım hızlı kanal kinetiği yüksek çözünürlüklü Xenopus oositlerde eksprese edilen voltaj-bağımlı iyon kanallarının iyonik ve yolluk akımlarının düşük ses kayıtları elde etmek için kullanılır. Küçük değişiklikler ile, voltaj kıskaç fluorometri kesme açık oosit protokolüne akuple edilebilir. 

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

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

Xenopus laevis Oosit, Mikroenjeksiyon

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Biyoloji

Fertilization of Xenopus oocytes using the Host Transfer Method

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as Xenopus and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In Xenopus, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host's oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.

Fertilization of Xenopus oocytes using the Host Transfer Method

Procedure for fertilizing Xenopus oocytes by the host transfer method.

Gübrelenmesi Xenopus oositleri Host Transfer Yöntemi kullanılarak

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to ...

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

Fare Oosit Mikroenjeksiyon, olgunlaşması ve ploidi Değerlendirmesi

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

Single Oocyte Bisulfite Mutagenesis

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for regulating gene transcription1. DNA methylation is an epigenetic modification that acts predominantly as a repressive mark. Through the covalent addition of a methyl group onto cytosines in CpG dinucleotides, it can recruit additional repressive proteins and histone modifications to initiate processes involved in condensing chromatin and silencing genes2. DNA methylation is essential for normal development as it plays a critical role in developmental programming, cell differentiation, repression of retroviral elements, X-chromosome inactivation and genomic imprinting.
One of the most powerful methods for DNA methylation analysis is bisulfite mutagenesis. Sodium bisulfite is a DNA mutagen that deaminates cytosines into uracils. Following PCR amplification and sequencing, these conversion events are detected as thymines. Methylated cytosines are protected from deamination and thus remain as cytosines, enabling identification of DNA methylation at the individual nucleotide level3. Development of the bisulfite mutagenesis assay has advanced from those originally reported4-6 towards ones that are more sensitive and reproducible7. One key advancement was embedding smaller amounts of DNA in an agarose bead, thereby protecting DNA from the harsh bisulfite treatment8. This enabled methylation analysis to be performed on pools of oocytes and blastocyst-stage embryos9. The most sophisticated bisulfite mutagenesis protocol to date is for individual blastocyst-stage embryos10. However, since blastocysts have on average 64 cells (containing 120-720 pg of genomic DNA), this method is not efficacious for methylation studies on individual oocytes or cleavage-stage embryos.
Taking clues from agarose embedding of minute DNA amounts including oocytes11, here we present a method whereby oocytes are directly embedded in an agarose and lysis solution bead immediately following retrieval and removal of the zona pellucida from the oocyte. This enables us to bypass the two main challenges of single oocyte bisulfite mutagenesis: protecting a minute amount of DNA from degradation, and subsequent loss during the numerous protocol steps. Importantly, as data are obtained from single oocytes, the issue of PCR bias within pools is eliminated. Furthermore, inadvertent cumulus cell contamination is detectable by this method since any sample with more than one methylation pattern may be excluded from analysis12. This protocol provides an improved method for successful and reproducible analyses of DNA methylation at the single-cell level and is ideally suited for individual oocytes as well as cleavage-stage embryos.

Bisulfite mutagenesis is the gold standard for analyzing DNA methylation. Our modified protocol allows for DNA methylation analysis at the single-cell level and was specifically designed for individual oocytes. It can also be used for cleavage-stage embryos.

Tek Oosit Bisülfit Mutagenez

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for ...

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage checkpoint signaling as a surveillance mechanism to sense DNA damage and direct cellular responses to DNA damage. There are several groups of proteins called sensors, transducers and effectors involved in DNA damage checkpoint signaling (Figure 1). In this complex signaling pathway, ATR (ATM and Rad3-related) is one of the major kinases that can respond to DNA damage and replication stress. Activated ATR can phosphorylate its downstream substrates such as Chk1 (Checkpoint kinase 1). Consequently, phosphorylated and activated Chk1 leads to many downstream effects in the DNA damage checkpoint including cell cycle arrest, transcription activation, DNA damage repair, and apoptosis or senescence (Figure 1). When DNA is damaged, failing to activate the DNA damage checkpoint results in unrepaired damage and, subsequently, genomic instability. The study of the DNA damage checkpoint will elucidate how cells maintain genomic integrity and provide a better understanding of how human diseases, such as cancer, develop.
Xenopus laevis egg extracts are emerging as a powerful cell-free extract model system in DNA damage checkpoint research. Low-speed extract (LSE) was initially described by the Masui group1. The addition of demembranated sperm chromatin to LSE results in nuclei formation where DNA is replicated in a semiconservative fashion once per cell cycle.
The ATR/Chk1-mediated checkpoint signaling pathway is triggered by DNA damage or replication stress 2. Two methods are currently used to induce the DNA damage checkpoint: DNA damaging approaches and DNA damage-mimicking structures 3. DNA damage can be induced by ultraviolet (UV) irradiation, &gamma;-irradiation, methyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitroquinoline-1-oxide (4-NQO), or aphidicolin3, 4. MMS is an alkylating agent that inhibits DNA replication and activates the ATR/Chk1-mediated DNA damage checkpoint 4-7. UV irradiation also triggers the ATR/Chk1-dependent DNA damage checkpoint 8. The DNA damage-mimicking structure AT70 is an annealed complex of two oligonucleotides poly-(dA)70 and poly-(dT)70. The AT70 system was developed in Bill Dunphy's laboratory and is widely used to induce ATR/Chk1 checkpoint signaling 9-12.
Here, we describe protocols (1) to prepare cell-free egg extracts (LSE), (2) to treat Xenopus sperm chromatin with two different DNA damaging approaches (MMS and UV), (3) to prepare the DNA damage-mimicking structure AT70, and (4) to trigger the ATR/Chk1-mediated DNA damage checkpoint in LSE with damaged sperm chromatin or a DNA damage-mimicking structure.

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

Xenopus egg extract is a useful model system to investigate the DNA damage checkpoint. This protocol is for the preparation of Xenopus egg extracts and DNA damage checkpoint inducing reagents. These techniques are adaptable to a variety of DNA damaging approaches in the study of the DNA damage checkpoint signaling.

Kullanılarak DNA hasar Checkpoint incelenmesi<em&gt; Xenopus</em&gt; Yumurta ayıklar

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage ...

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Spermatozoa are male reproductive cells especially designed to reach, recognize and fuse with the egg. To perform these tasks, sperm cells must be prepared to face a constantly changing environment and to overcome several physical barriers. Being in essence transcriptionally and translationally silent, these motile cells rely profoundly on diverse signaling mechanisms to orient themselves and swim in a directed fashion, and to contend with challenging environmental conditions during their journey to find the egg. In particular, Ca2+-mediated signaling is pivotal for several sperm functions: activation of motility, capacitation (a complex process that prepares sperm for the acrosome reaction) and the acrosome reaction (an exocytotic event that allows sperm-egg fusion). The use of fluorescent dyes to track intracellular fluctuations of this ion is of remarkable importance due to their ease of application, sensitivity, and versatility of detection. Using one single dye-loading protocol we utilize four different fluorometric techniques to monitor sperm Ca2+ dynamics. Each technique provides distinct information that enables spatial and/or temporal resolution, generating data both at single cell and cell population levels.

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Intracellular Ca2+ dynamics are very important in sperm physiology and Ca2+-sensitive fluorescent dyes constitute a versatile tool to study them. Population experiments (fluorometry and stopped flow fluorometry) and single cell experiments (flow cytometry and single cell imaging) are used to track spatio-temporal [Ca2+] changes in human sperm cells.

Hücre içi Ca Ölçme<sup&gt; 2 +</supDört Teknikleri ile İnsan rm&gt; Değişiklikler: Konvansiyonel Florometre, Akış Florometre, Sitometrisi ve Tek Hücre Görüntüleme Durduruldu

Spermatozoa  are male reproductive cells especially designed to reach, recognize and fuse  with the egg. To perform these tasks, sperm cells must be ...

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including by mechanical forces. Certain TRPV4 mutations cause complex hereditary bone or neuronal pathologies in human. Wild-type or mutant TRPV4 transgenes are commonly expressed in cultured mammalian cells and examined by Fura-2 fluorometry and by electrodes. In terms of the mechanism of mechanosensitivity and the molecular bases of the diseases, the current literature is confusing and controversial. To complement existing methods, we describe two additional methods to examine the molecular properties of TRPV4. (1) Rat TRPV4 and an aequorin transgene are transformed into budding yeast. A hypo-osmtic shock of the transformant population yields a luminometric signal due to the combination of aequorin with Ca2+, released through the TRPV4 channel. Here TRPV4 is isolated from its usual mammalian partner proteins and reveals its own mechanosensitivity. (2) cRNA of TRPV4 is injected into Xenopus oocytes. After a suitable period of incubation, the macroscopic TRPV4 current is examined with a two-electrode voltage clamp. The current rise upon removal of inert osmoticum from the oocyte bath is indicative of mechanosensitivity. The microAmpere (10-6 to 10-4 A) currents from oocytes are much larger than the subnano- to nanoAmpere (10-10 to 10-9 A) currents from cultured cells, yielding clearer quantifications and more confident assessments. Microscopic currents reflecting the activities of individual channel proteins can also be directly registered under a patch clamp, in on-cell or excised mode. The same oocyte provides multiple patch samples, allowing better data replication. Suctions applied to the patches can activate TRPV4 to directly assess mechanosensitivity. These methods should also be useful in the study of other types of TRP channels.

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

To complement commonly used methods to study TRPV4&#8217;s, two methods are described: Its mechanosensitivity can be studied by Ca2+-aequorin luminometry in transgenic yeast upon hypo-osmotic challenge. It can also be examined in TRPV4-RNA injected Xenopus oocytes by whole-cell two-electrode voltage clamp or patch clamp in on-cell or excised mode.

Maya lüminometrik ve<em&gt; Xenopus</emTRPV4 Moleküler Mechanosensitivity arasında&gt; Yumurta Elektrofizyolojik Sınavları

TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Ksenopus Translation Düşüklüğü Belirlemek için Bir Model Olarak laevis

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

3D-FRAP Mikroskopisi ile Gap Birleşimine Bağlı MiRNA Transferinin Analizi

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular mechanisms behind the beneficial effects of exercise are poorly understood. Many physical workouts, particularly those classified as aerobic exercises such as jogging and walking, produce impulsive forces at the time of foot contact with the ground. Therefore, it was speculated that mechanical impact might be implicated in how exercise contributes to organismal homeostasis. For testing this hypothesis on the brain, a custom-designed ''passive head motion'' (hereafter referred to as PHM) system was developed that can generate vertical accelerations with controlled and defined magnitudes and modes and reproduce mechanical stimulation that might be applied to the heads of rodents during treadmill running at moderate velocities, a typical intervention to test the effects of exercise in animals. By using this system, it was demonstrated that PHM recapitulates the serotonin (5-hydroxytryptamine, hereafter referred to as 5-HT) receptor subtype 2A (5-HT2A) signaling in the prefrontal cortex (PFC) neurons of mice. This work provides detailed protocols for applying PHM and measuring its resultant mechanical accelerations at rodents' heads.

The present protocol describes a custom-designed ''passive head motion'' system, which reproduces mechanical accelerations at rodents' heads generated during their treadmill running at moderate velocities. It allows dissecting mechanical factors/elements from the beneficial effects of physical exercise.

Kemirgenlerin Kafalarında Tanımlanmış İvmelenmeler Oluşturmak için Pasif Kafa Hareketinin Uygulanması

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...