This work was supported by NIH Grants R01-DC010146 and R01-DC010397. Its content is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health.

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The authors declare no existing or potential conflict of interest.

في هذا التقرير وصفنا كيفية الثقافة خلايا التعليم العالي-OC1 واستخدامها لتقييم آليات للخلايا المخدرات التي يسببها والتحقيق في الخصائص الفنية للprestin، المحرك الجزيئي للقوقعة OHCs. الإجراءات الفنية، ومع ذلك، فإن العام بما فيه الكفاية لتتكيف بسهولة مع مختلف الدراسات. <p cl...

منزل الأذن معهد-الجهاز كورتي 1 تستمد (HEI-OC1) خلايا من الجهاز السمعي من 1،2 الماوس المعدلة وراثيا. حضانة أي خلية من هذا الفأر وراثيا في 33 ° C / 10٪ CO 2 (شروط متساهلة) يستحث تعبير عن الجينات تخليد الذي يقوم بتشغيل دي التمايز وتسارع انتشار. تحريك الخلايا إلى 39 ° C / 5٪ CO 2 (ظروف غير متساهلة) تؤدي إلى انخفاض انتشار، والتمايز، وعلى الأقل في حالة من مؤسسات التعليم العالي-OC1، موت الخلايا 2،3. تم استنساخ خلايا التعليم العالي-OC1 وتميزت في المختبر لدينا منذ أكثر من عشر سنوات، وأشارت الدراسات الأولية أنها تعبير عن علامات معينة من خلايا الشعر قوقعة، مثل prestin، الميوسين 7A، Atoh1، عامل التغذية العصبية، calbindin وكالمودولين، ولكن أيضا علامات من دعم خلايا مثل connexin 26 و نمو الخلايا الليفية مستقبلات عامل (جمعية جيل المستقبل-R) 2. لذلك، اقترح أن مؤسسات التعليم العالي-OC1 يمكن أن تمثل commoن السلف للخلايا الحسية ودعم من جهاز كورتي 2. قدمت دراسات موازية أدلة قوية على أن التوراتية المخدرات السامة مثل سيسبلاتين، جنتاميسين والستربتومايسين التي يسببها كاسباس 3 التنشيط في هذه الخلايا، في حين لم المخدرات تعتبر غير سامة للأذن، مثل البنسلين، وليس 2،3. لذلك، اقترح هذا الخط الخلية كنظام في المختبر للتحقيق في الآليات الخلوية والجزيئية تشارك في تسميم أذني وللفحص من احتمال التسمم الأذني أو خصائص otoprotective العقاقير الدوائية الجديدة. وتشير التقديرات إلى أن الخلايا مؤسسات التعليم العالي-OC1 استخدمت في أكثر من مائة وخمسين دراسة نشرت في السنوات العشر الماضية. في حين تبحث في التأثير المحتمل الموالية لأفكارك من الأدوية المختلفة كان الهدف الرئيسي لمعظم الدراسات التي تنطوي على هذا الخط الخلية، عمليات الخلية الهامة الأخرى مثل الالتهام الذاتي والشيخوخة قد بدأت لتوها إلى التحقيق في زنازين مؤسسات التعليم العالي-OC1 4-7. أنانا دراسة حديثة من مختبرنا 8، استخدمنا خلايا التعليم العالي-OC1 لجمع مجموعة شاملة من البيانات عن موت الخلايا والبقاء والانتشار، الشيخوخة والالتهام الذاتي الناجم عن العقاقير الدوائية المختلفة التي يكثر استخدامها في العيادة. نحن أيضا مقارنة بعض استجابات الخلايا مؤسسات التعليم العالي-OC1 مع تلك من كلوة-293 (خلايا الكلى الجنينية البشرية) وهيلا (خلايا الظهارية البشرية) يتلقون العلاج متطابقة. وأشارت النتائج التي توصلنا إليها خلايا التعليم العالي-OC1 تستجيب إلى كل دواء بطريقة مميزة، مع dose- مميزة والحساسية تعتمد على الوقت واحدة على الأقل من آليات قيد الدراسة. أكدنا أيضا في هذه الدراسة أن التفسير الصحيح للنتائج التجريبية سوف يتطلب إجراء دراسات موازية مع تقنية أكثر من 8. وفي دراسة مختلفة ونحن التحقيق في استخدام الخلايا مؤسسات التعليم العالي-OC1 لتقييم الاستجابة الوظيفية لprestin، البروتين السيارات من خلايا الشعر الخارجي قوقعة (OHCs) 9 </sتصل&gt;. أبلغنا التدفق الخلوي ودراسات المسح المجهري ليزر متحد البؤر على نمط من التعبير prestin، وكذلك السعة غير الخطية (NLC) والدراسات لقط خلية التصحيح كلها في الخلايا مؤسسات التعليم العالي-OC1 مثقف في متساهلة (P-HEI-OC1) وغير متساهلة (NP-HEI-OC1) الظروف. وأشارت النتائج التي توصلنا إليها كل من إجمالي التعبير prestin وغشاء البلازما زيادة توطين بطريقة تعتمد على الوقت في الخلايا NP-HEI-OC1. ومن المثير للاهتمام، وجدنا أيضا أن الزيادة في توطين prestin في الغشاء البلازمي للخلايا NP-HEI-OC1 المترابطة مع انخفاض في نا + K + أتباز، التي translocated من غشاء البلازما إلى السيتوبلازم دون تغييرات كبيرة في إجمالي التعبير الخلية. وبالإضافة إلى ذلك، أثبتنا أن الخلايا P-HEI-OC1 لها مؤتمر العمل النيجيري القوي المرتبطة prestin الوظائف الحركية، والتي انخفضت عندما زادت كثافة جزيئات prestin الحاضرة في غشاء البلازما. وإجمالا، فإن هذه النتائج تدعم بقوة فائدة مؤسسات التعليم العالي-OC1 خلايا للتحقيق البروتينات السمعية. في هذه المقالة الفيديو وصفنا كيفية الثقافة خلايا التعليم العالي-OC1، لماذا أنها مريحة لاستخدام خلايا تنمو في الظروف متساهلة (P-HEI-OC1) للدراسات السمية الخلوية، وكيفية تقييم آلية / ثانية للخلايا المخدرات التي يسببها وكيف إجراء دراسات الكهربية (على سبيل المثال، والتصحيح، المشبك، والسعة غير الخطية (NLC)) للتحقيق في الخصائص الفنية للprestin، المحرك الجزيئي للOHCs القوقعة.

<table border="0" cellpadding="0" cellspacing="0" width="1447">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:905px;">HEI-OC1 cells</td>
			<td style="width:143px;"></td>
			<td style="width:139px;"></td>
			<td style="width:260px;">ALL THE ASSAY KITS, EQUIPMENTS&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Class II Biological Safety cabinet</td>
			<td>The Baker Company</td>
			<td>Sterilgard III</td>
			<td>AND COMPANIES INDICATED IN THE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Refrigerated centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td>PREVIOUS 2 COLUMNS ARE ONLY</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inverted microscope</td>
			<td>Zeiss</td>
			<td>Axiovert 25</td>
			<td>EXAMPLES, AND ANY OTHER SIMILAR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Waterbath</td>
			<td>Stovall</td>
			<td>HWB115</td>
			<td>PRODUCT COULD BE USED.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell counter</td>
			<td>Nexcelom</td>
			<td>Cellometer Auto T4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Two (2) Cell incubators, one at 33&#176;C/10% CO2 and other at 39&#176;C/5% CO2</td>
			<td>Forma Scientific</td>
			<td>3110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell culture dishes, PS, 100 x 20 mm with vents</td>
			<td>Greinier Bio-One</td>
			<td>664-160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell culture dishes , PS,&#160; 60 x 15 mm with vents&#160;</td>
			<td>Greiner Bio-One</td>
			<td>628160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cellstar tissue culture flasks&#160; 250 mL</td>
			<td>Greiner Bio-One</td>
			<td>658-175</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cellstar tissue cultur&#160; flasks 550 mL</td>
			<td>Greiner Bio-One</td>
			<td>660-175</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;6 well cell culture plate, with lid-Cellstar</td>
			<td>Greiner Bio-One</td>
			<td>657-160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microtest Tissue culture plate, 96 well,flat bottom with lid</td>
			<td>Becton Dickinson</td>
			<td>353072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro-Assay-Plate, Chimmey, 96-well white,clear botton</td>
			<td>Greiner Bio-One</td>
			<td>655098</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 ml Polypropylene conical tube with cap Cellstars</td>
			<td>Becton Dickinson&#160;</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 ml Polypropylene conical tubes with cap-Cellstars</td>
			<td>Greiner Bio-One</td>
			<td>188-271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS pH 7.4 (1X)&#160;</td>
			<td>Life Technologies</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)</td>
			<td>Life Technologies</td>
			<td>11965-084</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum (FBS)&#160;</td>
			<td>Hyclone</td>
			<td>SH10073.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leibovitz&#39;s L-15 Medium, no phenol red</td>
			<td>Gibco/Invitrogen</td>
			<td>21083-027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin, 0.25%&#160;</td>
			<td>Life Technologies</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TACS MTT Cell Proliferation Assay Kit</td>
			<td>Trevigen</td>
			<td>4890-25-K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Caspase-Glo 3/7 Assay&#160; kit</td>
			<td>Promega</td>
			<td style="width:139px;">&#160; G8091&#160;</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BrdU Cell Proliferation Assay Kit&#160;</td>
			<td>Cell Signaling</td>
			<td>6813</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non-enzymatic cell dissociation solution&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C5789</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell-Tox Green Cytotoxicity Assay Kit</td>
			<td>Promega</td>
			<td style="width:139px;">&#160; G8741&#160;</td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACSAriaIII instrument&#160;</td>
			<td>BD Biosciences</td>
			<td>&#160;FACSAriaIII</td>
			<td>With 488 nm excitation (blue laser)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digital Blot Scanner</td>
			<td>LI-COR</td>
			<td>C-DiGit</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electrophoresis and Blotting Unit</td>
			<td>Hoefer</td>
			<td>SE300 miniVE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spectra Max 5 Plate Reader with Soft Max Pro 5.2 Software</td>
			<td>Molecular Devices</td>
			<td>SpectraMax 5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Patch-clamp amplifier</td>
			<td>HEKA</td>
			<td>EPC-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Puller for preparing patch electrodes</td>
			<td>Sutter Instruments</td>
			<td>P-97</td>
			<td></td>
		</tr>
	</tbody>
</table>

working with auditory hei-oc1 cells

HEI-OC1 is one of the few mouse auditory cell lines available for research purposes. Originally proposed as an in vitro system for screening of ototoxic drugs, these cells have been used to investigate drug-activated apoptotic pathways, autophagy, senescence, mechanism of cell protection, inflammatory responses, cell differentiation, genetic and epigenetic effects of pharmacological drugs, effects of hypoxia, oxidative and endoplasmic reticulum stress, and expression of molecular channels and receptors. Among other several important markers of cochlear hair cells, HEI-OC1 cells endogenously express prestin, the paradigmatic motor protein of outer hair cells. Thus, they can be very useful to elucidate novel functional aspects of this important auditory protein. HEI-OC1 cells are very robust, and their culture usually does not present big complications. However, they require some special conditions such as avoiding the use of common anti-bacterial cocktails containing streptomycin or other antibiotics as well as incubation at 33 &#176;C to stimulate cell proliferation and incubation at 39 &#176;C to trigger cell differentiation. Here, we describe how to culture HEI-OC1 cells and how to use them in some typical assays, such as cell proliferation, viability, death, autophagy and senescence, as well as how to perform patch-clamp and non-linear capacitance measurements.

في بضع من المنشورات الحديثة أبلغنا مجموعة شاملة من الدراسات التي تهدف إلى تقييم استجابة الخلايا مؤسسات التعليم العالي-OC1 لعدة عقاقير الدوائية التي يشيع استخدامها وكذلك التحقيق في وظيفة prestin 8،9. في هذه الدراسات التي قطعناها على أنفسنا استخدام ...

Watch this Scientific Journal Video about Working with Auditory HEI-OC1 Cells at JoVE.com

Working with Auditory HEI-OC1 Cells

House Ear Institute-Organ of Corti 1 (HEI-OC1) is one of the few mouse auditory cell lines currently available for research purposes. This protocol describes how to work with HEI-OC1 cells to investigate the cytotoxic effects of pharmacological drugs as well as functional properties of inner ear proteins.

العمل مع الخلايا السمعية مؤسسات التعليم العالي-OC1

HEI-OC1 is one of the few mouse auditory cell lines available for research purposes. Originally proposed as an in vitro system for screening of ototoxic ...

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

14.9K Views.  University of California, Los Angeles. House Ear Institute-Organ of Corti 1 (HEI-OC1) is one of the few mouse auditory cell lines currently available for research purposes. This protocol describes how to work with HEI-OC1 cells to investigate the cytotoxic effects of pharmacological drugs as well as functional properties of inner ear proteins.

Video: Working with Auditory HEI-OC1 Cells

Transfecting Human Neural Stem Cells with the Amaxa Nucleofector

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection reagents has often resulted in poor cell viability and low transfection efficiency.  Other mechanical methods of introducing a gene of interest, such as a &#8220;gene gun&#8221; or microinjection, are also limited by poor cell viability and low numbers of transfected cells.  The strategy of using viral constructs to introduce an exogenous gene into primary cells has been constrained by both the amount of time and labor required to create viral vectors and potential safety concerns.  We describe here a step-by-step protocol for transfecting hNSPCs using Amaxa's Nucleofector device and technology with electrical current parameters and buffer solutions specifically optimized for transfecting neural stem cells.  Using this protocol, we have achieved initial transfection efficiencies of ~35% and ~70% after stable transfection.  The protocol entails combining a high number of hNSPCs with the DNA to be transfected in the appropriate buffer followed by electroporation with the Nucleofector device.

Introducing a gene of interest into a cell is a powerful method for elucidating its function in vivo. This protocol describes an efficient method of transfecting a culture of human neural stem/precursor cells (hNSPCs) using the Nucleofector electroporation apparatus made by Amaxa.

Transfecting الإنسان الخلايا الجذعية العصبية مع Nucleofector Amaxa

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection ...

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.

وسم الخلايا الجذعية مع الأصباغ نيون للمنظمات غير الغازية مع الكشف عن التصوير الضوئي

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 ...

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass1. In this article, we apply this technique to image exocytosis of synaptic vesicles in retinal bipolar cells isolated from the goldfish retina. These neurons are very suitable for this kind of study due to their large axon terminals. By simultaneously patch clamping the bipolar cells, it is possible to investigate the relationship between pre-synaptic voltage and synaptic release2,3. Synaptic vesicles inside the bipolar cell terminals are loaded with a fluorescent dye (FM 1-43&reg;) by co-puffing the dye and a ringer solution containing a high K+ concentration onto the synaptic terminals. This depolarizes the cells and stimulates endocytosis and consequent dye uptake into the glutamatergic vesicles. After washing the excess dye away for around 30 minutes, cells are ready for being patch clamped and imaged simultaneously with a 488 nm laser. The patch pipette solution contains a rhodamine-based peptide that binds selectively to the synaptic ribbon protein RIBEYE4, thereby labeling ribbons specifically when terminals are imaged with a 561 nm laser. This allows the precise localization of active zones and the separation of synaptic from extra-synaptic events.

In this video, we demonstrate how to label and visualize single synaptic vesicle exocytosis and trafficking in goldfish retinal bipolar cells using total internal reflectance fluorescence (TIRF) microscopy.

إيماس التصوير في خلايا الشبكية مع القطبين الميكروسكوب TIRF

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective ...

Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent progress, the functional properties and lateralization of the human auditory cortex are far from being fully understood. Transcranial Magnetic Stimulation (TMS) is a non-invasive technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses, and represents a unique method of exploring plasticity and connectivity. It has only recently begun to be applied to understand auditory cortical function 2. 
An important issue in using TMS is that the physiological consequences of the stimulation are difficult to establish. Although many TMS studies make the implicit assumption that the area targeted by the coil is the area affected, this need not be the case, particularly for complex cognitive functions which depend on interactions across many brain regions 3. One solution to this problem is to combine TMS with functional Magnetic resonance imaging (fMRI). The idea here is that fMRI will provide an index of changes in brain activity associated with TMS. Thus, fMRI would give an independent means of assessing which areas are affected by TMS and how they are modulated 4. In addition, fMRI allows the assessment of functional connectivity, which represents a measure of the temporal coupling between distant regions. It can thus be useful not only to measure the net activity modulation induced by TMS in given locations, but also the degree to which the network properties are affected by TMS, via any observed changes in functional connectivity.
Different approaches exist to combine TMS and functional imaging according to the temporal order of the methods. Functional MRI can be applied before, during, after, or both before and after TMS. Recently, some studies interleaved TMS and fMRI in order to provide online mapping of the functional changes induced by TMS 5-7. However, this online combination has many technical problems, including the static artifacts resulting from the presence of the TMS coil in the scanner room, or the effects of TMS pulses on the process of MR image formation. But more importantly, the loud acoustic noise induced by TMS (increased compared with standard use because of the resonance of the scanner bore) and the increased TMS coil vibrations (caused by the strong mechanical forces due to the static magnetic field of the MR scanner) constitute a crucial problem when studying auditory processing. 
This is one reason why fMRI was carried out before and after TMS in the present study. Similar approaches have been used to target the motor cortex 8,9, premotor cortex 10, primary somatosensory cortex 11,12 and language-related areas 13, but so far no combined TMS-fMRI study has investigated the auditory cortex. The purpose of this article is to provide details concerning the protocol and considerations necessary to successfully combine these two neuroscientific tools to investigate auditory processing. 
Previously we showed that repetitive TMS (rTMS) at high and low frequencies (resp. 10 Hz and 1 Hz) applied over the auditory cortex modulated response time (RT) in a melody discrimination task 2. We also showed that RT modulation was correlated with functional connectivity in the auditory network assessed using fMRI: the higher the functional connectivity between left and right auditory cortices during task performance, the higher the facilitatory effect (i.e. decreased RT) observed with rTMS. However those findings were mainly correlational, as fMRI was performed before rTMS. Here, fMRI was carried out before and immediately after TMS to provide direct measures of the functional organization of the auditory cortex, and more specifically of the plastic reorganization of the auditory neural network occurring after the neural intervention provided by TMS. 
Combined fMRI and TMS applied over the auditory cortex should enable a better understanding of brain mechanisms of auditory processing, providing physiological information about functional effects of TMS. This knowledge could be useful for many cognitive neuroscience applications, as well as for optimizing therapeutic applications of TMS, particularly in auditory-related disorders.

Auditory processing is the basis of speech and music-related processing. Transcranial Magnetic Stimulation (TMS) has been used successfully to study cognitive, sensory and motor systems but has rarely been applied to audition. Here we investigated TMS combined with functional Magnetic Resonance Imaging to understand the functional organization of auditory cortex.

تعيين آثار ما بعد من ثيتا انفجار تحفيز على القشرة السمعية الإنسان مع التصوير الوظيفي

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

الاستقراء وتحليل الظهارية الوسيطة الانتقالية ل

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

كفاءة الجيل المستحثة الإنسان افرة القوة الخلايا الجذعية من الخلايا الجسدية البشرية مع سينداي الفيروسات

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

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.

التصوير مضان مع نانومتر دقة واحدة (FIONA)

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

Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been described, certain types of cells are resistant to many of these methods and yield low transfection efficiency1, potentially hindering research in those cell types. In this protocol, we present an optimized transfection procedure to introduce luciferase reporter genes as a plasmid DNA into the RAW264.7 macrophage cell line. Two different types of transfection reagents (lipid-based and polyamine-based) are described, and important notes are given throughout the protocol to ensure that the RAW264.7 cells are minimally altered by the transfection procedure and any experimental data obtained are the direct results of the experimental treatment. While transfection efficiency may not be higher compared to other transfection methods, the described procedure is robust enough for detecting luciferase signal in RAW264.7 without changing the physiological response of the cells to stimuli.

Transfection into the macrophage cell line, RAW264.7, is difficult due to the cell&#8217;s natural response against foreign materials. We described here a gentle yet robust procedure for transfecting luciferase reporter genes into RAW264.7 cells.

بنقل العدوى إلى خلايا RAW264.7 مع luciferase المراسل مراسل جين

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been ...

Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways that cannot be accomplished in vivo. For example, inotropic medications commonly also affect preload and afterload, precluding load-independent assessments of their myocardial effects in vivo. Additionally, this model allows for sampling of coronary sinus effluent without contamination from systemic venous return, permitting assessment of myocardial oxygen consumption. Further, the advent of miniaturized pressure-volume catheters has allowed for the precise quantification of markers of both systolic and diastolic performance. We describe a model in which the left ventricle can be studied while performing both volume and pressure work under controlled conditions. 
In this technique, the heart and lungs of a Sprague-Dawley rat (weight 300-500 g) are removed en bloc under general anesthesia. The aorta is dissected free and cannulated for retrograde perfusion with oxygenated Krebs buffer. The pulmonary arteries and veins are ligated and the lungs removed from the preparation. The left atrium is then incised and cannulated using a separate venous cannula, attached to a preload block. Once this is determined to be leak-free, the left heart is loaded and retrograde perfusion stopped, creating the working heart model. The pulmonary artery is incised and cannulated for collection of coronary effluent and determination of myocardial oxygen consumption. A pressure-volume catheter is placed into the left ventricle either retrograde or through apical puncture. If desired, atrial pacing wires can be placed for more precise control of heart rate. This model allows for precise control of preload (using a left atrial pressure block), afterload (using an afterload block), heart rate (using pacing wires) and oxygen tension (using oxygen mixtures within the perfusate).

Isolated working heart models can be used to measure the effect of loading conditions, heart rate, and medications on myocardial performance and oxygen consumption. We describe methods for preparation of a rodent left heart working model that permits study of systolic and diastolic performance and oxygen consumption under various conditions.

القوارض العمل نموذج القلب لدراسة أداء عضلة القلب واستهلاك الأوكسجين

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

سم كلوي حقن مكروي في الزرد لنموذج قصور كلوي حاد

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...