We thank Lowell Cochran for expert videography assistance. The financial assistance of the Canadian Institutes of Health Research (CIHR), the Canadian Breast Cancer Foundation (CBCF, Ontario Chapter), the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canadian Breast Cancer Research Alliance, the Ontario Centers of Excellence, the Breast Cancer Action Kingston and the Clare Nelson bequest fund through grants to LR is gratefully acknowledged. SG was the recipient of an NSERC studentship. MG was supported by a postdoctoral fellowship from the US Army Breast Cancer Program, the Ministry of Research and Innovation of the Province of Ontario and the Advisory Research Committee of Queen&#8217;s University.

1. طلاء الخلايا في Electroporation للغرف <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> في غطاء الصفحي التدفق، يعرض للتريبسين الخلايا باستخدام تقنية معقمة كالمعتاد. ملاحظة: من المهم جدا للقضاء بواسطة الطرد المركزي كل آثار التربسين، لأنها قد تعيق انتشار الخلايا على الزجاج، وبالتالي تشكيل الملتصقة وتقاطعات الفجوة. </li><li style=";text-align:right;direction:rtl"> ماصة 1 مل من تعليق خلية في غرف Electroporation للالمعقمة المتوفرة مع electroporator في الموقع (الشكل 1)، ومكان في 37 درجة مئوية، CO 2 الحاضنة. يمكن تحسين التصاق الخلية بواسطة طلاء على فبرونيكتين، والكولاجين، بولي يسين أو الخلية والأنسجة لاصقة (انظر الجدول المواد): ملاحظة. </li><li style=";text-align:right;direction:rtl"> عندما شكلت خلايا طبقة متموجة انهم مستعدون للفحص GJIC. </li></ol> 2. Electroporation للإجراء ويمكن إجراء فحص لElectroporation للGJIC خارج غطاء الصفحي التدفق،نظرا لأنه يستغرق سوى بضع دقائق. إذا كانت هناك حاجة مرات حضانة أطول لإجراء تجربة معينة، ومن ثم يمكن إجراء تماما في غطاء تدفق الصفحي. في جميع الحالات، يجب على الدوائر التي تزرع فيها الخلايا تكون عقيمة. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد 5 ملغ / مل حل لوسيفر الأصفر: يذاب 10 ملغ LY مسحوق (المتوفرة مع electroporator) في 2 مل خالية من الكالسيوم متوسطة النمو. للتجارب التي تتطلب مرات حضانة أطول، فلتر تعقيم الحل وتخزينها في 4 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> نضح المتوسطة النمو وغسل الخلايا مع خالية من الكالسيوم المتوسطة، والحرص على عدم خدش أحادي الطبقة أو تجف الخلايا. إذا تجفف الخلايا التي عادة ما يكون نوى أكثر قتامة والتقاط LY دون Electroporation لل. وأثر هو أكثر وضوحا في منتصف الشريحة التي تتعرض أكثر إلى مسودات الهواء (الشكل 4F). </li><li style=";text-align:right;direction:rtl"> مع pipettor إيبندورف، ماصة محلول الصبغة إلى الخلايا (400 ميكرولتر للغرفة بأكملها)، على حافة الغرفة، بeing الحرص على عدم لمس طبقة الخلايا. </li><li style=";text-align:right;direction:rtl"> وضع غرفة في حامل المرفق مع electroporator وتطبيق نبض لقوة المناسبة (انظر مناقشة). </li><li style=";text-align:right;direction:rtl"> نضح بعناية بعض من الحل LY. ويمكن إعادة استخدامها مرة ثانية للتجارب أقل أهمية. </li><li style=";text-align:right;direction:rtl"> إضافة خالية من الكالسيوم DMEM تحتوي على 10٪ مصل dialysed. </li><li style=";text-align:right;direction:rtl"> احتضان الخلايا لمدة 3-5 دقيقة في الحاضنة للسماح بنقل الصبغة من خلال تقاطعات الفجوة. ملاحظة: إدراج مصل dialysed في هذه المرحلة تساعد إغلاق المسام. </li><li style=";text-align:right;direction:rtl"> غسل الصبغة الفردية مع خالية من الكالسيوم DMEM للمراقبة الخلية الحية. بدلا من ذلك، وخلايا يمكن أن تكون ثابتة في هذه المرحلة، من خلال إضافة 4٪ فورمالدهايد إلى البئر، ثم تغسل مع PBS (الفوسفات مخزنة المالحة). في جميع الحالات يجب غسل إزالة كافة الخلفية. ملاحظة: في التجارب الأولية لتحديد الظروف المثلى، إذا كان الجهد منخفض جدا، فمن الممكن للسماح للخلايا التعافي في incubatoص لبضع دقائق وelectroporate نفس الشريحة للمرة الثانية، لتوفير في تكلفة الشرائح. </li></ol> 3. فحص مجهرية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> مراقبة الخلايا تحت إضاءة مضان، باستخدام مجهر مقلوب مجهزة المرشح المناسب للصبغة المستخدمة. لوسيفر الأصفر، الإثارة هو 423nm، 555nm الانبعاثات، فلتر WBV لمجهر نيكون IX70. </li><li style=";text-align:right;direction:rtl"> القضاء على الآثار الغضروف المفصلي عن طريق وضع غطاء زجاجي الانزلاق على رأس البلاستيك جيدا، بعد ملئه إلى الأعلى مع السائل، لدراسة مورفولوجيا الخلايا تحت النقيض من المرحلة. لأفضل الصور، وكذلك يمكن فصل من الشريحة، وساترة وضعت على الإطار. لخلايا ثابتة، وكذلك يمكن ملء مع الجلسرين للمراقبة مجهرية، في حين أن الخلايا الحية يجب أن تكون مليئة النمو المتوسطة. ملاحظة: الغسل والتصوير أسهل إذا تم إصلاح الخلايا مع الفورمالديهايد بعد نقل الصبغة (الخطوة 2.7 أعلاه). إذا كانت الخلايا لار الثابتة، ويتم القضاء على مضان من الخلايا داخل ما يقرب من 60 دقيقة وهذا يتوقف على نوع من الخلايا، والجهد وكمية LY أدخلت بينما في الخلايا الثابتة يتم الاحتفاظ مضان لعدة ساعات. ومع ذلك، يتلاشى مضان أو تبدد بعد O / N الحضانة، حتى في الخلايا الثابتة. لهذا السبب، يجب أن تؤخذ الصور قريبا بعد Electroporation لل(الشكل 2). </li></ol> 4. الكميات من الاتصالات بين الخلايا <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تصوير الخلايا مع الهدف 20X تحت مضان والنقيض من المرحلة (الشكل 3). </li><li style=";text-align:right;direction:rtl"> تحديد ووضع علامة مع نجم الخلايا electroporated على الحدود مع المنطقة غير electroporated (الشكل 2، السهم، Electroporation للحافة). </li><li style=";text-align:right;direction:rtl"> تحديد ووضع علامة مع نقطة خلايا الاستشعاع على الجانب غير electroporated، حيث قامت بتحويل الصبغة من خلال تقاطعات الفجوة (أرقام 3A و 3B). </li><li style=";text-align:right;direction:rtl"> تقسيم العدد الكلي للالاستشعاع الخلايا على منطقة غير electroporated من قبل عدد من خلايا electroporated على طول حافة (أرقام 3A و 3B). ملاحظة: يتم احتساب نقل من 200 على الأقل خلايا electroporated الحدود متجاورة لكل تجربة. عدد حصل هو القيمة GJIC. </li></ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+gap+junctional+intercellular+communication+based+on+digital+image+analysis.">Quantification of gap junctional intercellular communication based on digital image analysis.</a> Am J Physiol Regul Integr Comp Physiol. 297 (2), R243-R247 (2009).</li><li>Raptis, L., Lamfrom, H., Benjamin, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cellular+phenotype+and+expression+of+polyomavirus+middle+T+antigen+in+rat+fibroblasts.">Regulation of cellular phenotype and expression of polyomavirus middle T antigen in rat fibroblasts.</a> Mol Cell Biol. 5, 2476-2486 (1985).</li><li>Raptis, L., Marcellus, R. C., Whitfield, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+membrane-associated+protein+kinase+C+activity+correlates+to+tumorigenicity+but+not+anchorage-independence+in+a+clone+of+mouse+NIH+3T3+cells.">High membrane-associated protein kinase C activity correlates to tumorigenicity but not anchorage-independence in a clone of mouse NIH 3T3 cells.</a> Exp Cell Res. 207, 152-154 (1993).</li><li>Vultur, A., Cao, J., Arulanandam, R., Turkson, J., Jove, R., Greer, P., Craig, A., Elliott, B. E., Raptis, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+to+cell+adhesion+modulates+Stat3+activity+in+normal+and+breast+carcinoma+cells.">Cell to cell adhesion modulates Stat3 activity in normal and breast carcinoma cells.</a> Oncogene. 23, 2600-2616 (2004).</li><li>Vultur, A., Arulanandam, R., Turkson, J., Niu, G., Jove, R., Raptis, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stat3+is+required+for+full+neoplastic+transformation+by+the+Simian+Virus+40+Large+Tumor+antigen.">Stat3 is required for full neoplastic transformation by the Simian Virus 40 Large Tumor antigen.</a> Mol Biol Cell. 16, 3832-3846 (2005).</li></ol>

The corresponding author is the inventor in a patent held by Queen&#8217;s University, which has been licensed to Cell Projects Inc. The company or the University had no influence upon the contents of the paper.

الخطوات الحاسمة في البروتوكول المواد Electroporated نقاء المواد التي سيتم electroporated مهم جدا. إذا كانت الخلايا مسطحة جدا، ثم تركيزات أعلى من تتبع صبغة يجب استخدام (تصل إلى 20 ملغ / مل لLY) وفي مثل هذه الحالات، والنقاء هو أكثر أهمية...

تطبيق التيار الكهربائي إلى خلية يسبب تشكيل المسام على غشاء الخلية، من خلال عملية تسمى Electroporation لل. مسام تسمح بمرور مجموعة متنوعة من الجزيئات nonpermeant من خلال الغشاء. يمكن التحكم في المجال الكهربائي على وجه التحديد، بحيث المسام شكلت صغيرة جدا وreclose بسرعة، مع الحد الأدنى من اضطراب في الفيزيولوجيا الخلوية. ومن المثير للاهتمام، الخلايا الملتصقة يمكن زراعتها على شريحة زجاجية مغطاة موصل وشفافة أكسيد الإنديوم والقصدير (ايتو) وelectroporated في الموقع، وهذا هو على السطح حيث تنمو، دون أن يتم فصل electroporated في تعليق. خلايا يمكن أن تنمو بشكل جيد جدا على هذا السطح، وبما أنها تابعة والموسعة، الملاحظة المجهرية مفصلة ممكن. باستخدام هذه التقنية، جزيئات صغيرة nonpermeant يمكن إدخال الفور وبشكل أساسي إلى 100٪ من الخلايا، مما يجعل هذه التقنية مناسبة خاصة بالنسبة للدراسات حول تفعيل شركاتonents من مسار التالية التحفيز يجند لمستقبلات (مراجعة في 1). وقد استخدم Electroporation للفي الغالب لإدخال الحمض النووي (وتسمى أيضا electrotransfection). ومع ذلك، يمكن Electroporation للفي الموقع أن تكون ذات قيمة لإدخال مجموعة كبيرة ومتنوعة من الجزيئات، مثل الببتيدات 2-4، أليغنوكليوتيد]، مثل العقاقير RNA، المزدوج تقطعت بهم السبل الحمض النووي [أليغنوكليوتيد شرك لمنع عامل النسخ ملزمة، أو سيرنا 3،5، 6، 7-10 النيوكليوتيدات المشعة، والبروتينات 11 12 أو الموالية للمخدرات 13. بعد Electroporation لل، والخلايا هي lysed يمكن للالتحليلات الكيميائية الحيوية أو ثابتة وملطخة الأجسام المضادة. طلاء ايتو موصل رقيقة جدا، 800-1،000Å، بحيث لا تشعر بالانزعاج نمو الخلايا بواسطة الفرق في ارتفاع السطوح اثنين، كما تنمو الخلايا عبر حافة طلاء موصل. وهذا يوفر ميزة أنه غير electrop-خلايا orated يمكن زراعتها جنبا إلى جنب مع تلك electroporated، لتكون بمثابة الضوابط. يمكن استخدام نفس النهج لفحص موصلي الفجوة والاتصال بين الخلايا (GJIC)، كما هو موضح في الفيديو. تقاطعات الفجوة هي القنوات التي تربط الداخلية من الخلايا المجاورة 14. الفجوة موصلي والاتصال بين الخلايا تلعب دورا هاما في تشكيل الورم والانبثاث، في حين أن الجينات المسرطنة مثل SRC قمع GJIC 15،16. لدراسة GJIC صبغة الفلورسنت مثل لوسيفر الأصفر (LY) غالبا ما أدخلت الخلايا المستزرعة من خلال حقن مكروي أو كشط التحميل 17 و لوحظ انتشار الصبغة في الخلايا المجاورة مجهريا تحت إضاءة مضان. لكن هذه التقنيات دائما يسبب تلف الخلايا. وصفنا الآن تقنية حيث تزرع الخلايا على شريحة زجاجية التي وهي مغلفة جزئيا مع ايتو 18. تم تطبيق نبضة كهربائية في وجود LY (أو الأصباغ الأخرى) كاليفورنياباستخدام انتشارها في الخلايا المتزايد من جانب موصل من الشريحة، في حين صبغ الهجرة إلى الخلايا المجاورة، غير electroporated، وقد لوحظ من خلال المجهر مضان الإضاءة. لتجنب الخلايا الحساسة المزعجة التي قد تميل إلى فصل من أحادي الطبقة، جمعية صمم التي لا تتطلب إلكترود لتوضع على رأس من الخلايا لتطبيق الحالي 19 الكهربائي. يقدم هذا النهج القدرة ل quantitate GJIC في عدد كبير من الخلايا، ودون أي اضطراب يمكن كشفها لالأيض الخلوي، كما يدل على ذلك عدم وجود تأثير على طول المرحلة G1 التالية التحفيز المصل 12، وزيادة في مستويات منظمات المزارعين البروتين protooncogene (Raptis، غير منشورة) أو اثنين تحركات المرتبطة بالإجهاد الخلوي، خنزير P38 أو JNK / SAPK كيناز 1. هذا النهج جعل من الممكن فحص الارتباط بين مستويات الجين الورمي التعبير والتحول وGJIC 18، فضلا عن تأثير SRC وSTAT3 على GJIC في مجموعة متنوعة من أنواع الخلايا، بما فيها الخلايا المستزرعة حديثا من عينات ورم الرئة 20-23. بالإضافة إلى ذلك، Electroporation للالموقعي مع الإعداد الذي وصفه تفتقر إلى القطب العلوي واستخدمت بنجاح لمظاهرة من إغلاق الفجوة تقاطع على التمايز adipocytic، على الرغم من التعلق الخلوي إلى الركيزة يتم تخفيض في تلك المرحلة في 19،24.

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			<td height="21" style="height:21px;width:179px;">DMEM</td>
			<td style="width:428px;">Cellgro http://www.cellgro.com/</td>
			<td style="width:119px;">50-013-PB</td>
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			<td height="21" style="height:21px;width:179px;">DMEM without Calcium</td>
			<td style="width:428px;">Hyclone (Thermo scientific: http://www.thermoscientific.com)</td>
			<td style="width:119px;">SH30319-01</td>
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			<td height="21" style="height:21px;width:179px;">Donor Calf Serum&#160;</td>
			<td style="width:428px;">PAA: http://www.paa.com/</td>
			<td style="width:119px;">Cat.# B15-008</td>
			<td></td>
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			<td height="21" style="height:21px;width:179px;">Fetal Bovine Serum</td>
			<td style="width:428px;">PAA: http://www.paa.com/</td>
			<td style="width:119px;">Cat.# A15-751</td>
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			<td height="21" style="height:21px;width:179px;">Electroporation apparatus</td>
			<td style="width:428px;">Cell Projects Ltd UK: http://www.cellprojects.com/</td>
			<td style="width:119px;">ACE-100</td>
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			<td height="21" style="height:21px;width:179px;">Chambers</td>
			<td style="width:428px;">Cell Projects Ltd UK</td>
			<td style="width:119px;">ACE-04-CC</td>
			<td>4-wells</td>
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			<td height="21" style="height:21px;width:179px;">Chambers</td>
			<td style="width:428px;">Cell Projects Ltd UK</td>
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			<td>8-wells</td>
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			<td height="21" style="height:21px;width:179px;">Lucifer Yellow</td>
			<td style="width:428px;">Cell Projects Ltd UK</td>
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			<td>High purity</td>
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			<td style="width:428px;">BD Biosciences</td>
			<td style="width:119px;">354240</td>
			<td>Cell and tissue adhesive</td>
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			<td height="21" style="height:21px;width:179px;">Fibronectin</td>
			<td style="width:428px;">Sigma Aldrich</td>
			<td style="width:119px;">F1141</td>
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			<td height="21" style="height:21px;width:179px;">Collagen</td>
			<td style="width:428px;">BD Biosciences</td>
			<td style="width:119px;">354236</td>
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			<td height="21" style="height:21px;width:179px;">Poly-Lysine</td>
			<td style="width:428px;">Sigma Aldrich</td>
			<td style="width:119px;">P8920</td>
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a functional assay for gap junctional examination; electroporation of adherent cells on indium-tin oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.&#160; Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive&#160;glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.

ويبين الشكل 2 كبد الفئران خلايا الظهارية T51B 22، electroporated مع LY وتصويرها تحت مضان (لوحة A و B)، أو على النقيض من المرحلة (C) الإضاءة، وبعد التثبيت والغسيل. في لوحة A، يتم وضع علامة على حافة منطقة الجزاء electroporated باللون الأحمر. التدرج من مضان على يمين الخط الأحمر ?...

Watch this Scientific Journal Video about A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide at JoVE.com

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

مقايسة الوظيفية للفجوة صلي فحص. Electroporation للمن الخلايا الملتصقة على الإنديوم أكسيد القصدير

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. ...

a-functional-assay-for-gap-junctional-examination-electroporation

Research

JoVE Journal

Biology

9.9K Views.  Queen's University. This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

Video: A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

Patterning Cells on Optically Transparent Indium Tin Oxide Electrodes

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular constructs serving as in vitro mimics of native tissues.  In this study, photolithography and wet etching techniques were used to fabricate individually addressable indium tin oxide (ITO) electrodes on glass substrates.  The glass substrates containing ITO microelectrodes were modified with poly(ethylene glycol) (PEG) silane to make them protein and cell resistive.  Presence of insulating PEG molecules on the electrode surface was verified by cyclic voltammetry employing potassium ferricyanide as a redox reporter molecule.  Importantly, the application of reductive potential caused desorption of the PEG layer, resulting in regeneration of the conductive electrode surface and appearance of typical ferricyanide redox peaks.    Application of reductive potential also corresponded to switching of ITO electrode properties from cell non-adhesive to cell-adhesive. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.  Micropatterning of several cell types was demonstrated on these substrates.   In the future, the control of the biointerfacial properties afforded by this method will allow to engineer cellular microenvironments through the assembly of three or more cell types into a precise geometric configuration on an optically transparent substrate.

Non-fouling PEG silane monolayer was desorbed from individually addressable ITO electrodes on glass by application of a reductive potential. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.

الزخرفة على خلايا شفافة بصريا أقطاب إنديوم أكسيد القصدير

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular ...

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.

ووضع العلامات hESCs hMSCs مع أوكسيد الحديد النانوية لعدم الغازية في تعقب المجراة مع التصوير بالرنين المغناطيسي

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

Electroporation of Mycobacteria

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall, but is compounded by the fact that most molecular techniques have been developed for distantly-related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified and DNA transformation of many mycobacterial species have now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the  conditions for the electroporation pulse. Optimized methods for the transformation of both slow- and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.

Mycobacterial pathogenic strategies remain poorly understood. The slow growth rate of most species, the impenetrable nature of the cell-wall, and the hazards of working with pathogens make mycobacteria difficult to study and are largely responsible for our poor understanding of these organisms. In this video we will demonstrate the technique of electroporation, which involves subjecting cells to a brief high electrical impulse to allow the entry of DNA. It is the most widely used method for introducing DNA into mycobacterial cells.

Electroporation من مايكوباكتيريا

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly ...

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

Electroporation في الجسم الحي وخارج الرحم للتعبير الجينات في خلايا الشبكية العقدة ماوس

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

في تقطير التحت خلوية الموقع من خلايا الثدييات وغير ملتصقة منتسب -

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

الطفرات والتحليل الوظيفي للقنوات ايون أعربت Heterologously في خلايا الثدييات

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

مولد الرمع الفحص : خلايا منتسب

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific shapes. Drosophila tracheal terminal cells have become a powerful genetic model to identify and elucidate the roles of genes required for generating cellular morphologies. Terminal cells are a component of a branched tubular network, the tracheal system that functions to supply oxygen to internal tissues. Terminal cells are an excellent model for investigating questions of cell shape as they possess two distinct cellular architectures. First, terminal cells have an elaborate branched morphology, similar to complex neurons; second, terminal cell branches are formed as thin tubes and contain a membrane-bound intracellular lumen. Quantitative analysis of terminal cell branch number, branch organization and individual branch shape, can be used to provide information about the role of specific genetic mechanisms in the making of a branched cell. Analysis of tube formation in these cells can reveal conserved mechanisms of tubulogenesis common to other tubular networks, such as the vertebrate vasculature. Here we describe techniques that can be used to rapidly fix, image, and analyze both branching patterns and tube formation in terminal cells within Drosophila larvae. These techniques can be used to analyze terminal cells in wild-type and mutant animals, or genetic mosaics. Because of the high efficiency of this protocol, it is also well suited for genetic, RNAi-based, or drug screens in the Drosophila tracheal system.

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Here, we present a method for light microscopy analysis of tracheal terminal cells in Drosophila larvae. This method allows for quick examination of branch and lumen morphology in whole animals and would be useful for analysis of individual mutants or screens for mutations affecting terminal cell development.

فحص<em&gt; ذبابة الفاكهة</em&gt; اليرقات الخلايا الطرفية الرغامي بواسطة المجهر الضوئي

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

قياس وتحليل حمض خارج الخلية الإنتاج لتحديد معدل حال السكر

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

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 ...