The Satchi-Fainaro research laboratory is partially supported by The Association for International Cancer Research (AICR), German-Israel Foundation (GIF), THE ISRAEL SCIENCE FOUNDATION (Grant No. 1309/10), Swiss Bridge Award, and by grants from the Israeli National Nanotechnology Initiative (INNI), Focal Technology Area (FTA) program: Nanomedicine for Personalized Theranostics, and by The Leona M. and Harry B. Helmsley Nanotechnology Research Fund.

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+SIKVAV+site+of+laminin+in+promotion+of+angiogenesis+and+tumor+growth:+an+in+vivo+Matrigel+model.">Role of the SIKVAV site of laminin in promotion of angiogenesis and tumor growth: an in vivo Matrigel model.</a> J Natl Cancer Inst. 84, 1633-1638 (1992).</li><li>Passaniti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple,+quantitative+method+for+assessing+angiogenesis+and+antiangiogenic+agents+using+reconstituted+basement+membrane,+heparin,+and+fibroblast+growth+factor.">A simple, quantitative method for assessing angiogenesis and antiangiogenic agents using reconstituted basement membrane, heparin, and fibroblast growth factor.</a> Lab Invest. 67, 519-528 (1992).</li><li>Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+models+of+angiogenesis.">In vivo models of angiogenesis.</a> J Cell Mol Med. 10, 588-612 (2006).</li><li>Auerbach, R., Lewis, R., Shinners, B., Kubai, L., Akhtar, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+assays:+a+critical+overview.">Angiogenesis assays: a critical overview.</a> Clin Chem. 49, 32-40 (2003).</li><li>Johns, A., Freay, A. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbubbles+in+medical+imaging:+current+applications+and+future+directions.">Microbubbles in medical imaging: current applications and future directions.</a> Nat Rev Drug Discov. 3, 527-532 (2004).</li><li>Kuliszewski, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+of+endothelial+progenitor+cell+engraftment+using+contrast-enhanced+ultrasound+and+targeted+microbubbles.">Molecular imaging of endothelial progenitor cell engraftment using contrast-enhanced ultrasound and targeted microbubbles.</a> Cardiovasc Res. 83, 653-662 (2009).</li><li>Stieger, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound+assessment+of+angiogenesis+in+a+matrigel+model+in+rats.">Ultrasound assessment of angiogenesis in a matrigel model in rats.</a> Ultrasound Med Biol. 32, 673-681 (2006).</li><li>Erez, N., Truitt, M., Olson, P., Arron, S. T., Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-Associated+Fibroblasts+Are+Activated+in+Incipient+Neoplasia+to+Orchestrate+Tumor-Promoting+Inflammation+in+an+NF-kappaB-Dependent.">Cancer-Associated Fibroblasts Are Activated in Incipient Neoplasia to Orchestrate Tumor-Promoting Inflammation in an NF-kappaB-Dependent.</a> Manner. 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The authors have nothing to disclose.

الجمع بين أساليب التصوير التكميلية يسمح تحصيل معلومات عن microvessel كثافة، وظائف الأوعية الدموية مكونات مختلفة &quot;والتشكل. CM تحميل على المقابس هلام ECM تقليد يولد المكروية الورم، التي لا يفصلها عن السكان الخلية الأخرى، مثل الخلايا البطانية، أن يتم تجنيد ويتسرب إلى المكو...

ووصف الأوعية الدموية matrigel المكونات فحص أول مرة من قبل Kibbey وآخرون في عام 1992، حيث كان يستخدم لتقييم تحفيز الأوعية الدموية التي SIKVAV الببتيد (سر إيل-ليز-فال-علاء-فال) 1. وعلى النقيض من غيرها من فحوصات الأوعية الدموية في الجسم الحي، مثل الأوعية الدموية القرنية الماوس أو فرخ المقايسات غشاء سقائية مشيمائية، هذا الاختبار هو سهل نسبيا لأداء 2. قد تحتوي على حقن ECM تقليد هلام الخلايا، الموالية للعائية أو المركبات المضادة للعائية و / أو عوامل. عند تقييم نشاط مركب المضادة للعائية، والمكونات يحتوي عادة على مزيج من العوامل الوعائية البطانية النمو (VEGF)، وعامل نمو الخلايا الليفية (جمعية جيل المستقبل)، ويمكن أن تدار على مادة مضادة للعائية مباشرة في المكونات أو جهازيا 3، 4. يتم حقن هلام ECM تقليد تحت الجلد حيث يتصلب في غضون دقائق. تقييم التوظيف الأوعية الدموية في قابس الناتج هو typicallذ يؤديها قياس مستويات الهيموغلوبين ضمن المكونات، من خلال قياس إشارة مضان بعد الحقن من ثيوسيانات فلوريسئين (FITC) -labeled ديكستران، من خلال المناعية من أقسام علم الأنسجة لعلامات البطانية محددة أو عن طريق خلية مضان تنشيط الفرز (FACS) 2،5، 6. ومع ذلك، هذا التقييم يسمح فقط تحليل نقطة النهاية ويفتقر إلى المعلومات حول الوظائف ومورفولوجية الأوعية الدموية. وبالإضافة إلى ذلك، قياسات حجم البلازما، إما عن طريق مستويات الهيموجلوبين أو باستخدام ديكستران-FITC كمؤشر، قد تكون مضللة إذ يتأثر المحتوى الدم عن طريق حجم الأوعية الدموية ومدى البرك الراكدة من الدم. هذا أمر بالغ الأهمية خصوصا عندما يتميز الأوعية الدموية تجنيده من قبل نفاذية تعزيز والاستبقاء (EPR) تأثير 7. نحن هنا اقتراح جديد أكثر دقة، طريقة لرؤية الأوعية الدموية تجنيدهم من خلال الجمع بين اثنين من طرائق التصوير التكميلية. HIGmicrobubbles ح-قرار تعزيز التباين الموجات فوق الصوتية (US) جنبا إلى جنب مع حيوي داخلي fibred-متحد البؤر endomicroscopy يمكن أن توفر المعلومات ليس فقط عن كثافة الأوعية الدموية، ولكن أيضا على التشكل والوظيفة. وعلاوة على ذلك، لا يمكن أن يؤديها هذا التحليل في عدة نقاط الوقت وبالتالي تمكين رصد حركية تكوين الأوعية الدموية. الولايات المتحدة هي طريقة التصوير المستخدمة على نطاق واسع التي تمتلك قرار مكانية عالية لتصوير الأوعية الدموية التالية في الوريد (IV) حقن microbubbles التي تبقى حصرا في مقصورة الأوعية الدموية 8-11. وmicrobubbles هم microbubbles مملوءة بالغاز التي تنتج إشارة مولد للصدى قوية عندما متحمس مع نبض الولايات المتحدة، وبالتالي تكون بمثابة عامل تباين جيد. يتم الحصول على صور 3D من المكونات باستخدام برنامج نظام التصوير الولايات المتحدة في أعقاب الدمار microbubbles. وتتكون الصور الناتجة عن إطارات صورة قبل وبعد التدمير من microbubbles ويعكس الاختلاف في كثافة الفيديو في اللون. هذه يغطىيتم عرض الصور تلقائيا من قبل البرنامج. ونتيجة لذلك، يمكن كميا في المئة من السفن وظيفية ضمن المكونات. عالية الدقة فيبيريد endomicroscopy متحد البؤر بمثابة طريقة التصوير التكميلية من خلال الإبلاغ عن الأوعية الدموية التشكل. في هذا الأسلوب مينيملي، يتم الحصول على الصور التالية الادارة الرابع من ديكستران FITC مترافق 12. البوليمر الفلورسنت الأصباغ مجرى الدم، وبالتالي بمثابة &quot;عامل تباين&quot; لالأوعية الدموية التشكل وتدفق الدم. وعلاوة على ذلك، منذ البوليمر حقن هو في حجم 70 كيلو دالتون، يمكن أن عبور السفن فقط راشح كما وجدت في ورم الأوعية الدموية. وهذا يؤدي إلى خلفية عالية من ديكستران FITC المسمى خارج الأوعية الدموية. ونتيجة لذلك، فيبيريد endomicroscopy متحد البؤر يمكن استخدامها لتصور الخصائص النموذجية للتأثير EPR في الورم neovasculature- الموسع، تتسرب منها المياه، والأوعية متشابكة للغاية، مع نهايات حادة. تم descri هذا نموذج النظامالسرير في مقارنة بين إمكانات عائية من U-87 MG ورم أرومي دبقي البشري واستنساخ نائمة لها (13). تم تقييم الأوعية الدموية داخل المقابس ثلاثة أسابيع بعد ECM تقليد هلام التلقيح. وقد تبين أن الأوعية الدموية داخل المقابس التي تحتوي CM من U-87 MG تم زيادة الخلايا المولدة للورم كبير من حيث العدد والكثافة وظيفة، بالمقارنة مع الأوعية الدموية داخل المقابس التي تحتوي CM من الخلايا المولدة للورم نائمة سريعة النمو. وبالتالي، كانت قادرة على استنتاج أن CM معزولة من المؤلفين U-87 MG سريعة النمو الخلايا المولدة للورم يحتوي على مستويات أعلى من العوامل المؤيدة للعائية، مقارنة مع CM من الخلايا المولدة للورم نائمة. هذه العوامل تحفز تشكيل الأوعية الدموية وظيفية عن طريق التأثير إيجابيا جميع الخطوات من تتالي عائية (أي انتشار، تبرعم، والهجرة، وأخيرا، وتشكيل هياكل أنبوبية من الخلايا البطانية).

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			<td dir="LTR" height="40" style="height:40px;width:255px;">Name of the Material/Equipment</td>
			<td dir="LTR" style="width:179px;">Company</td>
			<td dir="LTR" style="width:179px;">Catalog number</td>
			<td dir="LTR" style="width:251px;">Comments/Description&#160;</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:255px;">Rotary Vacuum Evaporator</td>
			<td dir="LTR" style="width:179px;">-</td>
			<td dir="LTR" style="width:179px;">-</td>
			<td style="width:251px;">-</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:255px;">Growth-factors reduced phenol-free Matrigel</td>
			<td dir="LTR" style="width:179px;">BD Bioscience</td>
			<td dir="LTR" style="width:179px;">FAL356231</td>
			<td style="width:251px;">Thaw overnight, at 40C on ice&#160;</td>
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		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:255px;">Depilatory cream&#160;</td>
			<td dir="LTR" style="width:179px;">-</td>
			<td dir="LTR" style="width:179px;">-</td>
			<td dir="LTR" style="width:251px;">-</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:255px;">Vevo2100 US imaging system</td>
			<td dir="LTR" style="width:179px;">VisualSonics Inc.&#160;</td>
			<td dir="LTR" style="width:179px;">-</td>
			<td dir="LTR" style="width:251px;">Requires previous knowledge in operating or the assistance of an authorized technician</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:255px;">55 MHz 708 probe</td>
			<td dir="LTR" style="width:179px;">VisualSonics Inc.</td>
			<td dir="LTR" style="width:179px;">-</td>
			<td dir="LTR" style="width:251px;">-</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:255px;">Vevo2100 imaging software</td>
			<td dir="LTR" style="width:179px;">VisualSonics Inc.</td>
			<td dir="LTR" style="width:179px;">-</td>
			<td dir="LTR" style="width:251px;">-</td>
		</tr>
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			<td dir="LTR" height="40" style="height:40px;width:255px;">VialMix&#160;</td>
			<td dir="LTR" style="width:179px;">DEFINITY Imaging</td>
			<td dir="LTR" style="width:179px;">VMIX</td>
			<td dir="LTR" style="width:251px;">-</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:255px;">DEFINITY microbubbles</td>
			<td dir="LTR" style="width:179px;">DEFINITY Imaging</td>
			<td dir="LTR" style="width:179px;">DE4</td>
			<td dir="LTR" style="width:251px;">Activate for 45 seconds using Vialmix before use</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:255px;">CellVizio (Fibered confocal endomicroscope)</td>
			<td dir="LTR" style="width:179px;">Mauna Kea Technologies</td>
			<td dir="LTR" style="width:179px;">-</td>
			<td dir="LTR" style="width:251px;">Requires previous knowledge in operating or the assistance of an authorized technician</td>
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		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:255px;">ProFlex MiniO/30 probe</td>
			<td dir="LTR" style="width:179px;">Mauna Kea Technologies</td>
			<td dir="LTR" style="width:179px;">-</td>
			<td dir="LTR" style="width:251px;">-</td>
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			<td dir="LTR" height="40" style="height:40px;width:255px;">70 kD FITC-labeled Dextran</td>
			<td dir="LTR" style="width:179px;">Sigma-Aldrich</td>
			<td dir="LTR" style="width:179px;">46945</td>
			<td dir="LTR" style="width:251px;">-</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:255px;">ImageCell software</td>
			<td dir="LTR" style="width:179px;">Mauna Kea Technologies</td>
			<td dir="LTR" style="width:179px;">-</td>
			<td dir="LTR" style="width:251px;">-</td>
		</tr>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:255px;">Calibration Kit</td>
			<td dir="LTR" style="width:179px;">Mauna Kea Technologies</td>
			<td dir="LTR" style="width:179px;">-</td>
			<td dir="LTR" style="width:251px;">-</td>
		</tr>
		<tr height="40">
			<td align="left" height="40" style="height:40px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td align="left">Gibco Life Tecchnologies</td>
			<td dir="LTR" style="width:179px;">41965-039</td>
			<td></td>
		</tr>
		<tr height="37">
			<td align="left" height="37" style="height:37px;">Fetal bovine serum (FBS)</td>
			<td align="left">Biological Industries&#160;</td>
			<td align="left">04-007-1A</td>
			<td></td>
		</tr>
		<tr height="39">
			<td align="left" height="39" style="height:39px;">Penicillin-Streptomycin-Nystatin Solution</td>
			<td align="left">Biological Industries&#160;</td>
			<td align="left">03-032-1B</td>
			<td></td>
		</tr>
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monitoring functionality and morphology of vasculature recruited by factors secreted by fast-growing tumor-generating cells

وmatrigel تحت الجلد المكونات الفحص في الفئران هو الأسلوب المفضل لفي التقييم في الجسم الحي من العوامل المؤيدة والمضادة للعائية. في هذه الطريقة، يتم إدخال العوامل المطلوبة في السائل البارد ECM تقليد الجل الذي، بعد الحقن تحت الجلد، يتصلب لتشكيل بيئة محاكاة الوسط السرطان. هذه المصفوفة تسمح تغلغل الخلايا المضيفة، مثل الخلايا البطانية، وبالتالي، وتشكيل الأوعية الدموية. وهنا نقترح تعديل مقايسة matrigel المكونات الجديدة التي يمكن استغلالها لتوضيح إمكانات عائية من مجموعة من العوامل التي يفرزها الخلايا السرطانية، بدلا من عامل معين (على سبيل المثال، bFGF وVEGF) أو وكيل. ويستخدم المكونات التي تحتوي على ECM تقليد هلام لتقديم المضيف (أي الماوس) مع مجموعة من العوامل يفرز إلى CM خلايا ورم أرومي دبقي مدرة للورم سريعة النمو. وصفناها سابقا المقارنة واسعة من إمكانيات عائية منU-87 MG ورم أرومي دبقي البشري واستنساخ لها نائمة المشتقة، في هذا النموذج نظام، والتي تبين الأوعية الدموية التي يسببها في U-87 خلايا MG الأبوية. تم إعداد CM من خلال تصفية وسائل الإعلام التي تم جمعها من لوحات زراعة الأنسجة متكدسة إما خط الخلية التالية 48 ساعة الحضانة. وبالتالي، فإنه يحتوي على العوامل الوحيدة التي يفرزها الخلايا، دون الخلايا نفسها. الموصوفة هنا هو مزيج من اثنين من طرائق التصوير، microbubbles محسنة النقيض التصوير بالموجات فوق الصوتية وحيوي داخلي-فيبيريد متحد البؤر endomicroscopy، لدقيقة، في الوقت الحقيقي توصيف ما، مورفولوجيا وظائف الأوعية الدموية التي شكلت حديثا داخل المقابس.

زرع سدادات هلام ECM مع تقليد CM من النمو السريع عائية خط الخلية المولدة للورم U-87 MG، والنتائج في الأوعية الدموية واسعة النطاق. هذه الأوعية الدموية يمكن استكشافها على نطاق واسع باستخدام طريقتين التصوير التكميلية. Microbubbles التصوير الول...

Watch this Scientific Journal Video about Monitoring Functionality and Morphology of Vasculature Recruited by Factors Secreted by Fast-growing Tumor-generating Cells at JoVE.com

Monitoring Functionality and Morphology of Vasculature Recruited by Factors Secreted by Fast-growing Tumor-generating Cells

We describe a matrigel plug assay to illustrate angiogenic potential of a pool of factors secreted by cancer cells using two complementary imaging modalities, ultrasound and endomicroscopy. The matrigel, an extracellular matrix (ECM)-mimic gel, is utilized to introduce the host (mouse) with angiogenic factors secreted to the conditioned media (C.M.).&#160;

مراقبة وظائف والصرف من الأوعية الدموية تجنيده من قبل العوامل التي يفرزها الخلايا المولدة للورم النمو السريع

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired ...

monitoring-functionality-morphology-vasculature-recruited-factors

Research

JoVE Journal

Biology

9.7K Views.  Tel Aviv University. We describe a matrigel plug assay to illustrate angiogenic potential of a pool of factors secreted by cancer cells using two complementary imaging modalities, ultrasound and endomicroscopy. The matrigel, an extracellular matrix (ECM)-mimic gel, is utilized to introduce the host (mouse) with angiogenic factors secreted to the conditioned media (C.M.). 

Video: Monitoring Functionality and Morphology of Vasculature Recruited by Factors Secreted by Fast-growing Tumor-generating Cells

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

اشتقاق الخلايا الجذعية الجنينية البشرية التي Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Monitoring Acupuncture Effects on Human Brain by fMRI

Functional MRI is used to study the effects of acupuncture on the BOLD response and the functional connectivity of the human brain.  Results demonstrate that acupuncture mobilizes a limbic-paralimbic-neocortical network and its anti-correlated sensorimotor/paralimbic network at multiple levels of the brain and that the hemodynamic response is influenced by the psychophysical response.  Physiological monitoring may be performed to explore the peripheral response of the autonomic nerve function. This video describes the studies performed at LI4 (hegu), ST36 (zusanli) and LV3 (taichong), classical acupoints that are commonly used for modulatory and pain-reducing actions. Some issues that require attention in the applications of fMRI to acupuncture investigation are noted.

FMRI and physiological monitoring is used to study the effects of Acupuncture on the central and peripheral nervous systems. Acupuncture mobilizes a limbic-paralimbic-neocortical network, with great overlap with the default mode network, to modulate neurological activity, possibly related to its autonomic effect in the peripheral nervous system.

رصد تأثيرات الوخز بالإبر في الدماغ بواسطة الرنين المغناطيسي الوظيفي الإنسان

Functional MRI is used to study the effects of acupuncture on the BOLD response and the functional connectivity of the human brain.  Results demonstrate ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

رسم مصير الخلايا الجذعية الجنينية البشرية من خلال تكوين ورم مسخي

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure 1 in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (&tilde;100 &mu;m) within the cells so that the SECs act as a sieve in which most chylomicrons, chylomicron remnants and macromolecules, but not cells, pass through to the hepatocytes and HSCs 1 (Fig. 1). Due to the lack of a basement membrane, the gap between the SECs and hepatocytes form the Space of Disse. HSCs occupy this space and play a prominent role in regulation and response to injury, storage of retinoic acid and immunoregulation of the liver 2.
SECs are among the most endocytically active cells of the body displaying an array of scavenger receptors on their cell surface 3. These include SR-A, Stabilin-1 and Stabilin-2. Generally, small colloidal particles less than 230 nm and macromolecules in buffer phase are taken up by SECs, whereas, large particles and cellular debris is endocytosed (phagocytosed) by KCs 4. Thus, the bulk clearance of extracellular material such as the glycosaminoglycans from blood is largely dependent on the health and endocytic functions of SECs 5,6. For example, an increase in blood hyaluronan levels is indicative of liver disease ranging from mild to more severe forms 7.
With the exception of one report 8, there are no immortalized SEC cell lines in existence. Even this immortalized cell line is de-differentiated in that it does not express scavenger receptors that are present on primary SECs (our data, not shown). All cell biological studies must be performed on primary cells obtained freshly from the animal. Unfortunately, SECs dedifferentiate under standard culture conditions and must be used within 1 or 2 days upon isolation from the animal. Differentiation of SECs is marked by the expression of Stabilin-2 or HARE receptor 9 , CD31, and the presence of cytoplasmic fenestration 1. Differentiation of SECs can be extended by the addition of VEGF in culture media or by culturing cells in hepatocyte conditioned medium 10,11.
In this report, we will demonstrate the endocytic activity of SECs in the intact organ using radio-labeled heparin for hyaluronan for the SEC-specific Stabilin-2 receptor. We will then purify hepatocytes and SECs from the perfused liver to measure endocytosis.

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The study of liver sinusoidal endothelial cells (SECs) must be performed with primary cells obtained from the animal as no cell lines exist. This method relies on liver digestion and differential centrifugation for SEC purification for subsequent culturing and experimentation.

في الإلتقام الكبد المجراة تلاه تنقية خلايا الكبد الكبد الإرواء

The liver is the metabolic center of the mammalian body and serves as a filter for the blood.  The basic architecture of the liver is illustrated in ...

Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting

RNA molecules play an essential role in biology. In addition to transmitting genetic information, RNA can fold into unique tertiary structures fulfilling a specific biologic role as regulator, binder or catalyst. Information about tertiary contact formation is essential to understand the function of RNA molecules. Hydroxyl radicals (&bull;OH) are unique probes of the structure of nucleic acids due to their high reactivity and small size.1 When used as a footprinting probe, hydroxyl radicals map the solvent accessible surface of the phosphodiester backbone of DNA1 and RNA2 with as fine as single nucleotide resolution. Hydroxyl radical footprinting can be used to identify the nucleotides within an intermolecular contact surface, e.g. in DNA-protein1 and RNA-protein complexes. Equilibrium3 and kinetic4 transitions can be determined by conducting hydroxyl radical footprinting as a function of a solution variable or time, respectively. A key feature of footprinting is that limited exposure to the probe (e.g., 'single-hit kinetics') results in the uniform sampling of each nucleotide of the polymer.5
In this video article, we use the P4-P6 domain of the Tetrahymena ribozyme to illustrate RNA sample preparation and the determination of a Mg(II)-mediated folding isotherms. We describe the use of the well known hydroxyl radical footprinting protocol that requires H2O2 (we call this the 'peroxidative' protocol) and a valuable, but not widely known, alternative that uses naturally dissolved O2 (we call this the 'oxidative' protocol). An overview of the data reduction, transformation and analysis procedures is presented.

Monitoring Equilibrium Changes in RNA Structure by &#039;Peroxidative&#039; and &#039;Oxidative&#039; Hydroxyl Radical Footprinting

This protocol describes how to quantify the Mg(II)-dependent formation of RNA tertiary structure by two methods of hydroxyl radical footprinting.

التغييرات في هيكل رصد التوازن الحمض النووي الريبي ل &#39;Peroxidative&#39; و &#39;مؤكسد&#39; Footprinting الهيدروكسيل

RNA molecules play an essential role in biology. In addition to transmitting genetic information, RNA can fold into unique tertiary structures fulfilling ...

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

رصد كيناز والفوسفاتيز الأنشطة من خلال دورة الخلية التي Ratiometric الحنق

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies 1-3. These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres 4-8.
Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell.
Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest 9. The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed.
This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to daughter cells. Ideal cells are adherent, easily transfected, and have large nuclei. This technique has been used to determine that 84% of EBV-derived plasmids are synthesized each generation and 88% of the newly synthesized plasmids partition faithfully to daughter cells in HeLa cells. Pairs of these EBV plasmids were seen to be tethered to or associated with sister chromatids after their synthesis in S-phase until they were seen to separate as the sister chromatids separated in Anaphase10. The method is currently being used to study replication of KSHV genomes in HeLa cells and SLK cells. HeLa cells are immortalized human epithelial cells, and SLK cells are immortalized human endothelial cells. Though SLK cells were originally derived from a KSHV lesion, neither the HeLa nor SLK cell line naturally harbors KSHV genomes11. In addition to studying viral replication, this visualization technique can be used to investigate the effects of the addition, removal, or mutation of various DNA sequence elements on synthesis, localization, and partitioning of other recombinant plasmid DNAs.

A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.

رصد النسخ المتماثل البلازميد في خلايا الثدييات يعيش أكثر من أجيال متعددة من مضان المجهري

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors are offering new opportunities for regenerative medicine. Such clinical applications of iPS cells have been limited so far, mainly due to the poor efficiency of the existing reprogramming methodologies and the risk of the generated iPS cells to form tumors upon implantation.
We hypothesized that the reprogramming of somatic cells towards pluripotency could be achieved in vivo by gene transfer of reprogramming factors. In order to efficiently reprogram cells in vivo, high levels of the Yamanaka (OKSM) transcription factors need to be expressed at the target tissue. This can be achieved by using different viral or nonviral gene vectors depending on the target tissue. In this particular study, hydrodynamic tail-vein (HTV) injection of plasmid DNA was used to deliver the OKSM factors to mouse hepatocytes. This provided proof-of-evidence of in vivo reprogramming of adult, somatic cells towards a pluripotent state with high efficiency and fast kinetics. Furthermore no tumor or teratoma formation was observed in situ.
It can be concluded that reprogramming somatic cells in vivo may offer a potential approach to induce enhanced pluripotency rapidly, efficiently, and safely compared to in vitro performed protocols and can be applied to different tissue types in the future.

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

This study demonstrates the reprogramming of somatic cells towards pluripotency in vivo without the generation of teratomas. We used hydrodynamic tail vein injection of plasmid DNA encoding the Yamanka factors to induce the in vivo reprogramming of adult hepatocytes into cells of enhanced pluripotency.

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors ...

Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains

Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a population always have accumulated mutations that are either detrimental or beneficial for the cell. If the fitness effect of a mutation provides the subpopulation with a strong selective growth advantage, the individuals of this subpopulation may rapidly outcompete and even completely eliminate their immediate fellows. Thus, small genetic changes and selection-driven accumulation of cells that have acquired beneficial mutations may lead to a complete shift of the genotype of a cell population. Here we present a procedure to monitor the rapid clonal expansion and elimination of beneficial and detrimental mutations, respectively, in a bacterial cell population over time by cocultivation of fluorescently labeled individuals of the Gram-positive model bacterium Bacillus subtilis. The method is easy to perform and very illustrative to display intraspecies competition among the individuals in a bacterial cell population.

Bacteria may accumulate either detrimental or beneficial mutations during their lifetime. In a population of cells individuals that have accumulated beneficial mutations may rapidly outcompete their fellows. Here we present a simple procedure to visualize intraspecies competition in a bacterial cell population over time using fluorescently labeled individuals.&#160;

مراقبة المنافسة داخل الأنواع في مجموعة الخلايا البكتيرية عن طريق Cocultivation من السلالات المسمى Fluorescently

Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

في فيفو تتبع 4 الأبعاد للالجذعية المكونة للدم والسلف الكبار في خلايا الفأر قبي نخاع العظم

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...