البحثية التي تدعمها جائزة جبهة الخلاص الوطني MCB-1021384.

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solvent+and+primary+deuterium+isotope+effects+show+that+lactate+CH+and+OH+bond+cleavage+are+concerted+in+Y254F+flavocytochrome+b2,+consistent+with+a+hydride+transfer+mechanism.">Solvent and primary deuterium isotope effects show that lactate CH and OH bond cleavage are concerted in Y254F flavocytochrome b2, consistent with a hydride transfer mechanism.</a> Biochemistry. 42, 15208-15214 (2003).</li><li>Kumar, S., Gawandi, V. B., Capito, N., Phillips, R. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=(&#956;-1,2-Peroxo)diiron(III/III)+complex+as+a+precursor+to+the+diiron(III/IV)+intermediate+X+in+the+assembly+of+the+iron&#45;radical+cofactor+of+ribonucleotide+reductase+from+mouse.">(&#956;-1,2-Peroxo)diiron(III/III) complex as a precursor to the diiron(III/IV) intermediate X in the assembly of the iron&#45;radical cofactor of ribonucleotide reductase from mouse.</a> Biochemistry. 46 (7), 1925-1932 (2007).</li><li>Pennati, A., Zanetti, G., Aliverti, A., Gadda, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+salt+and+pH+on+the+reductive+half-reaction+of+Mycobacterium+tuberculosis+FprA+with+NADPH+Biochemistry.">Effect of salt and pH on the reductive half-reaction of Mycobacterium tuberculosis FprA with NADPH Biochemistry.</a> Biochemistry. 47 (11), 3418-3425 (2008).</li><li>Patil, P. V., Allou, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+protocatechuate+dioxygenase+for+maintaining+anaerobic+conditions+in+biochemical+experiments.">The use of protocatechuate dioxygenase for maintaining anaerobic conditions in biochemical experiments.</a> Analytical Biochemistry. 286 (2), 187-192 (2000).</li></ol>

ليس لدينا ما يكشف.

الإنزيمات التي تحفز تفاعلات الأكسدة والاختزال عادة ما تحتوي على العوامل المساعدة مثل وhemes flavins أن تخضع لتغييرات كبيرة خلال الامتصاصية دورة الحفاز. شكل المؤكسد للفلافين يقدم ماكسيما الامتصاصية في ~ 360 و 450 نانومتر، وعادة ما يتم رصد الحد من قبل بعد انخفاض الامتصاصية في 450 نانومتر 7. بشكل عام، وبعض السلع الوسيطة عابر موجودة ولكن شكل والاضمحلال بسرعة جدا بحيث لا يمكن قياسها في مطياف العادية. باستخدام Photophysics التطبيقية SX20 توقف تدفق طيفي (أو أدوات مماثلة)، فمن الممكن لقياس التغيرات الامتصاصية في مقياس الوقت ميلي ثانية واحدة (ميت مرة، 2 مللي ثانية). درس هنا نحن في الاختزالية والاكسدة نصف ردود فعل monooxygenase فلافين التي تعتمد على وسيدا و، نموذجا يحتذى. وتم تحديد سعر نقل هيدريد عن طريق قياس التغير في الامتصاصية في 452 نانومتر بعد خلط انزيم مع NADPH في الظروف اللاهوائية. في وقت لاحق، مع الميزهوكان (ه) من وضع خلط مزدوج وثيقة وقف التدفق، كان رد فعل لأول مرة انزيم مع NADPH، حتى تم تحقيق تخفيض كامل، ثم كانت مختلطة في خفض انزيم NADP + مجمع مع الأكسجين. بعد هذا الإجراء، فإنه من الممكن الكشف عن عابر وسيطة فلافين الاوكسيجين وقياس معدلات تكوين والاضمحلال. وتحديد هذه السلع الوسيطة توفر البيانات التجريبية حول طبيعة هذا النوع رد فعل في الحفز. في حالة وجود سيدا و، تشكيل C4a-hydroperoxyflavin (رصدها عادة في 370-380 نانومتر)، والذي هو نوع hydroxylating. وبالإضافة إلى ذلك، وقياس معدل ثابت من كل خطوة واحد يسمح للحصول على معلومات عن خطوة تحديد سعر للتفاعل ويساعد على إلقاء الضوء على آليات الحركية والكيميائية للانزيم. بشكل عام، يمكن استخدام نهج مماثل لflavoenzymes أخرى، أو البروتينات التي الامتصاصية التغييرات التي حدثت، مثل البروتينات التي تابععين الهيم، بيريدكسال فوسفات، أو غير هيم الحديد 8-10. وجود قيود على هذا الأسلوب هو أن هناك حاجة إلى كميات كبيرة من انزيم المنقى، ولكن يمكن التغلب على ذلك عن طريق استخدام نظام التعبير ذات العوائد المرتفعة. واحد يحدد تركيز بروتين الأمثل لأطياف تسجيل عن طريق استخدام ما يكفي من البروتين بحيث يمكن ملاحظة إشارة قوية بما فيه الكفاية، ولكن ليس كثيرا حتى لا يضيع هذا الانزيم. عادة، فإن أقل تركيز انزيم لفلافين التي تحتوي على الانزيمات المستخدمة في توقف تدفق التجارب هو 6-10 ميكرومتر (بعد خلط) ويتم تحديده باستخدام المقابلة معامل الانقراض ضرس من الانزيم. في حالة وجود سيدا و، النسبة المئوية للFAD انزيم ملزمة هو 50-65٪ 2. ويعتبر الاشتقاق بروتين كما نشط في هذه التجارب لأن العامل المساعد FAD المنضم هو ضروري لالحفز. ومن أوجه القصور الممكنة لهذا الأسلوب هو في حالة العمليات في انزيم يحدث بشكل أسرع من 2 مللي ثانية (الوقت الميت من التدفق توقف) لن يكون لاحظوا، ولكن الوتفيد التقارير يحرث الاستراتيجيات التي يمكن أن انخفضت معدلات للتغلب على هذه المشكلة. مثال واحد لهذا يتضمن استخدام تركيزات عالية كلوريد الصوديوم في رد فعل على اختزال + NADP فيريدوكسين-11. لتنقية الأكسجين من الدائرة تدفق تدفق توقفت في كثير من الأحيان خطوة صعبة في هذه التجربة وتتطلب اهتماما خاصا. يستخدم الجلوكوز أوكسيديز الجلوكوز، نظام الموصوفة هنا بنجاح في معظم المختبرات كما هو وسيلة فعالة وغير مكلفة. ومع ذلك، هناك بعض السلبيات التي تشمل إنتاج H 2 O 2 وبالنسبة لبعض التطبيقات وينبغي النظر في بدائل أخرى مثل نظام دي أكسيجيناز-protocatechuate protocatechuate 12. الاستفادة من علبة القفازات اللاهوائية يجعل من الاسهل لضمان الظروف اللاهوائية، ولكن ليس ضروريا. ويجب إزالة الأكسجين من دائرة تدفق تدفق توقف لاننا نريد ان يتم تخفيض الانزيم في غياب الأكسجين أو تتفاعل مع الأكسجين في تركيزق الذي نحدده نحن. وعلى الرغم من توقف التدفق هو في علبة القفازات، وهناك أكسجين في دائرة تدفق إذا كنا مخازن الهوائية في التجارب السابقة. بالإضافة إلى قياسات الامتصاصية، يمكن تنفيذ المقايسات ازدواج اللون مضان والتعميم في معمل توقف تدفق للPhotophysics التطبيقية SX20 مع الملحقات ذات الصلة.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> عام معدات المختبرات </td><td> شركة </td><td> فهرس العدد </td></tr><tr><td> فراغ مضخة </td><td> وولش </td><td> - </td></tr><tr><td> بوخنر قوارير </td><td> الصياد </td><td> 70340-500 </td></tr><tr><td> إثارة القضبان </td><td> الصياد </td><td> 14-512-129 </td></tr><tr><td> إثارة لوحات </td><td> الصياد </td><td> 11-100-49S </td></tr><tr><td> شلنك خطوط </td><td> Kontes زجاج </td><td> - </td></tr><tr><td> الأرجون دبابة </td><td> Airgas </td><td> AR UPC300 </td></tr><tr><td> النيتروجين دبابة </td><td> Airgas </td><td> NI200 </td></tr><tr><td> خزان النيتروجين، ودرجة نقاء عالية جدا </td><td> Airgas </td><td> NI UHP200 </td></tr><tr><td> خزان الأكسجين </td><td> Airgas </td><td> OX 40 </td></tr><tr><td> 5 توازن النيتروجين خزان الهيدروجين٪ </td><td> Airgas </td><td> X02NI95B200H998 </td></tr><tr><td> SX20 القديسأوب تدفق طيفي </td><td> AppliedPhotophysics </td><td> - </td></tr><tr><td> علبة القفازات </td><td> خجول </td><td> - </td></tr><tr><td> ماء الحمام </td><td> BRINKMANN لاودا </td><td> - </td></tr><tr><td></td><td></td><td></td></tr><tr><td> لوازم </td><td></td><td></td></tr><tr><td> 50 مل BD الصقر أنابيب </td><td> الصياد </td><td> 14-432-23 </td></tr><tr><td> 15 مليلتر دينار بحريني الصقر مخروطي أنابيب </td><td> الصياد </td><td> 05-527-90 </td></tr><tr><td> 1.5 microcentrifuge إيبندورف أنابيب مل </td><td> الصياد </td><td> 05-402-18 </td></tr><tr><td> 50 و 25 قارورة زجاج مل </td><td> الصياد </td><td> 06-402 </td></tr><tr><td> سدادات المطاط </td><td> الصياد </td><td> 06-447H </td></tr><tr><td> الألومنيوم الاختام </td><td> الصياد </td><td> 06-406-15 </td></tr><tr><td></td><td></td><td></td></tr><tr><td> ReagenTS </td><td></td><td></td></tr><tr><td> فوسفات البوتاسيوم، أحادى </td><td> الصياد </td><td> AC2714080025 </td></tr><tr><td> فوسفات البوتاسيوم، ثنائي القاعدة </td><td> الصياد </td><td> P288-500 </td></tr><tr><td> خلات الصوديوم </td><td> سيغما </td><td> S-2889 </td></tr><tr><td> أوكسيديز الجلوكوز من أ. النيجر </td><td> سيغما </td><td> G7141-250KU </td></tr><tr><td> D-جلوكوز </td><td> الصياد </td><td> D16-500 </td></tr><tr><td> β-NADPH </td><td> الصياد </td><td> ICN10116783 </td></tr><tr><td> L (+)-الأورنيثين هيدروكلوريد </td><td> الصياد </td><td> ICN10116783 </td></tr></tbody></table>

monitoring the reductive and oxidative half-reactions of a flavin-dependent monooxygenase using stopped-flow spectrophotometry

الرشاشية الدخناء حاملة الحديد A (سيدا) هو monooxygenase FAD المحتوية على أن يحفز الهيدروكسيل من الأورنيثين في التركيب الحيوي من siderophores hydroxamate التي تعتبر ضرورية لالفوعة (على سبيل المثال ferricrocin أو N &#39;، N &quot;، N&#39;&#39;&#39;-triacetylfusarinine C) 1. ويمكن تقسيم رد فعل يحفزه سيدا إلى الاختزالية والأكسدة ردود الفعل نصف (مخطط 1). وفي التخفيض نصف رد فعل، يتم تقليل FAD تتأكسد منضما إلى سيدا و، بواسطة NADPH 2،3. وفي رد فعل الأكسدة نصف والعامل المساعد انخفاض يتفاعل مع الأوكسجين الجزيئي لتشكيل المتوسطة C4a-hydroperoxyflavin، والذي ينقل الأكسجين إلى ذرة الأورنيثين. هنا، ونحن تصف إجراء لقياس معدلات وكشف أشكال مختلفة من الأطياف سيدا باستخدام أداة توقف تدفق مثبتة في 1 علبة القفازات اللاهوائية. في صك توقف التدفق، كميات صغيرة من المواد الداخلة في التفاعل وبسرعة متفاوتة، وبعد أن تم إيقاف تدفق من قبل اله حقنة توقف (الشكل 1)، وتسجل التغيرات الطيفية من الحل وضعت في زنزانة الملاحظة مع مرور الوقت. في الجزء الأول من التجربة، وتبين لنا كيف يمكننا استخدام أداة توقف التدفق في وضع واحد، حيث يتم قياسها بشكل مباشر على تخفيض اللاهوائية من فلافين في وسيدا و بواسطة NADPH. نحن نستخدم ثم إعدادات خلط مزدوج حيث لا هوائيا اول خفض لسيدا و NADPH بواسطة لفترة محددة من الوقت في حلقة الشيخوخة، ومن ثم كان رد فعل مع الأكسجين الجزيئي في الخلية مراقبة (الشكل 1). من أجل تنفيذ هذه التجربة، ومخازن اللاهوائية ضرورية لأنه عندما يتم رصد سوى التخفيض نصف رد فعل، أي الأوكسجين في الحلول وتتفاعل مع العامل المساعد فلافين انخفاض وتشكيل وسيطة C4a-hydroperoxyflavin التي من شأنها أن تتحلل في نهاية المطاف العودة الى فلافين المؤكسد . فإن هذا لن تسمح للمستخدم لقياس بدقة معدلات انخفاض منذ لن يكون هناك دوران كامل للenzyme. عندما يجب أن تجرى حاليا دراسة الأكسدة نصف رد فعل يتم تخفيض الانزيم في غياب الأوكسجين بحيث يتم فقط لاحظت الخطوات بين الحد والأكسدة. واحد من المخازن المؤقتة المستخدمة في هذه التجربة هو الاوكسجين مشبعة حتى نتمكن من دراسة الأكسدة نصف رد فعل على تركيزات أعلى من الأوكسجين. هذه غالبا ما تكون الإجراءات المنفذة وعند دراسة إما الاختزالية أو مؤكسد نصف ردود فعل مع فلافين التي تحتوي على monooxygenases. مقياس الوقت من التجارب السابقة للحالة استقرار أداء مع التدفق توقف هو ميلي ثانية إلى ثانية، والتي تسمح بتحديد سعر الثوابت الجوهرية وكشف وتحديد وسيطة في رد فعل 4. ويمكن تطبيق الإجراءات المذكورة هنا لmonooxygenases فلافين التي تعتمد على الآخرين. 5،6

Watch this Scientific Journal Video about Monitoring the Reductive and Oxidative Half-Reactions of a Flavin-Dependent Monooxygenase using Stopped-Flow Spectrophotometry at JoVE.com

Monitoring the Reductive and Oxidative Half-Reactions of a Flavin-Dependent Monooxygenase using Stopped-Flow Spectrophotometry

وصفنا استخدام أداة توقف تدفق للتحقيق في كل من الاختزالية والاكسدة نصف ردود فعل<em&gt; الرشاشية الدخناء</em&gt; حاملة الحديد A (سيدا)، والتي تعتمد على monooxygenase فلافين. نعرض ثم الأطياف المقابلة لالأنواع في رد فعل من سيدا، ونحن حساب ثوابت معدل لتشكيلها.

رصد انتقاصية ومؤكسد نصف ردود فعل من Monooxygenase فلافين التي تعتمد على استخدام توقف تدفق الطيفي

Aspergillus fumigatus siderophore A (SidA) is an FAD-containing monooxygenase that catalyzes the hydroxylation of ornithine in the biosynthesis of ...

monitoring-reductive-oxidative-half-reactions-flavin-dependent

Research

JoVE Journal

Bioengineering

15.0K Views.  Virginia Polytechnic Institute and State University. وصفنا استخدام أداة توقف تدفق للتحقيق في كل من الاختزالية والاكسدة نصف ردود فعل الرشاشية الدخناء حاملة الحديد A (سيدا)، والتي تعتمد على monooxygenase فلافين. نعرض ثم الأطياف المقابلة لالأنواع في رد فعل من سيدا، ونحن حساب ثوابت معدل لتشكيلها.

Video: Monitoring the Reductive and Oxidative Half-Reactions of a Flavin-Dependent Monooxygenase using Stopped-Flow Spectrophotometry

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

عالية الإنتاجية مولد بروتين تعبير باستخدام منصة ميكروفلويديك

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Monitoring the Wall Mechanics During Stent Deployment in a Vessel

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the arterial wall lead to tissue injury, which initiates restenosis2-7. This hypothesis needs further investigations including better quantifications of non-uniform strain distribution on the artery following stent implantation. A non-contact surface strain measurement method for the stented artery is presented in this work. ARAMIS stereo optical surface strain measurement system uses two optical high speed cameras to capture the motion of each reference point, and resolve three dimensional strains over the deforming surface8,9. As a mesh stent is deployed into a latex vessel with a random contrasting pattern sprayed or drawn on its outer surface, the surface strain is recorded at every instant of the deformation. The calculated strain distributions can then be used to understand the local lesion response, validate the computational models, and formulate hypotheses for further in vivo study.

Stent-induced arterial strain distributions are characterized using an optical surface strain measurement system. This visualization technique is used to gain insights into the impact of stent implantation on the host vessel.

رصد ميكانيكا الحائط أثناء النشر ستينت في وعاء

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the ...

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

عدم الاتصال، تسمية خالية من رصد خلايا المصفوفة خارج الخلية، وباستخدام التحليل الطيفي رامان

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

الخلوي الجماعي: بروتوكول لضبط اليومية وعينات الخلايا تشغيل عداد الكريات الشامل على CyTOF

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

قياس الخواص الميكانيكية للخلايا الحية باستخدام المجهر القوة الذرية

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. Biosensors relying on the principle of FRET have enabled real-time visualization of subcellular signaling events in live cells with high temporal and spatial resolution. Here, we describe the application of a genetically encoded Bile Acid Sensor (BAS) that consists of two fluorophores fused to the farnesoid X receptor ligand binding domain (FXR-LBD), thereby forming a bile acid sensor that can be activated by a large number of bile acids species and other (synthetic) FXR ligands. This sensor can be targeted to different cellular compartments including the nucleus (NucleoBAS) and cytosol (CytoBAS) to measure bile acid concentrations locally. It allows rapid and simple quantitation of cellular bile acid influx, efflux and subcellular distribution of endogenous bile acids without the need for labeling with fluorescent tags or radionuclei. Furthermore, the BAS FRET sensors can be useful for monitoring FXR ligand binding. Finally, we show that this FRET biosensor can be combined with imaging of other spectrally distinct fluorophores. This allows for combined analysis of intracellular bile acid dynamics and i) localization and/or abundance of proteins of interest, or ii) intracellular signaling in a single cell.

We provide a detailed protocol to study bile acid dynamics in living cells using a genetically encoded BAS FRET sensor. This Bile Acid Sensor represents a unique tool to study (regulation of) bile acid transport and FXR activation in a wide range of cell types.

رصد في الوقت الحقيقي من بين الخلايا الصفراء حمض حيوية باستخدام حمض الصفراء الاستشعار القائمة على الحنق ترميز وراثيا

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

القسطرة الخفيفة داخل القلب للإشعال الجانبي لرصد الأيض الخلوي باستخدام مطياف الامتصاص عبر الجدارية لقلوب الثدييات المنصهرة

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between animal and clinical trials. Numerous animal and cell models have been used to examine the effects of drugs, yet these responses often differ in humans. Human cardiac slices offer an advantage for drug testing in that they are directly derived from viable human hearts. In addition to having preserved multicellular structures, cell-cell coupling, and extracellular matrix environments, human cardiac tissue slices can be used to directly test the effect of innumerable drugs on adult human cardiac physiology. What distinguishes this model from other heart preparations, such as whole hearts or wedges, is that slices can be subjected to longer-term culture. As such, cardiac slices allow for studying the acute as well as chronic effects of drugs. Furthermore, the ability to collect several hundred to a thousand slices from a single heart makes this a high-throughput model to test several drugs at varying concentrations and combinations with other drugs at the same time. Slices can be prepared from any given region of the heart. In this protocol, we describe the preparation of left ventricular slices by isolating tissue cubes from the left ventricular free wall and sectioning them into slices using a high precision vibrating microtome. These slices can then either be subjected to acute experiments to measure baseline cardiac electrophysiological function or cultured for chronic drug studies. This protocol also describes dual optical mapping of cardiac slices for simultaneous recordings of transmembrane potentials and intracellular calcium dynamics to determine the effects of the drugs being investigated.

This protocol describes the procedure for sectioning and culturing human cardiac slices for preclinical drug testing and details the use of optical mapping for recording transmembrane voltage and intracellular calcium signals simultaneously from these slices.

تقييم الكهربائيات القلبية قبل الاحتكين بواسطة الجهد المزدوج ورسم الخرائط البصرية الكالسيوم لشرائح القلب العضوية البشرية

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between ...

Imaging and Quantification of the Area of Fast-Moving Microbubbles Using a High-Speed Camera and Image Analysis

An experimental and image analysis technique is presented for imaging cavitation bubbles and calculating their area. The high-speed imaging experimental technique and image analysis protocol presented here can also be applied for imaging microscopic bubbles in other fields of research; therefore, it has a wide range of applications. We apply this to image cavitation around dental ultrasonic scalers. It is important to image cavitation to characterize it and to understand how it can be exploited for various applications. Cavitation occurring around dental ultrasonic scalers can be used as a novel method of dental plaque removal, which would be more effective and cause less damage than current periodontal therapy techniques. We present a method for imaging the cavitation bubble clouds occurring around dental ultrasonic scaler tips using a high-speed camera and a zoom lens. We also calculate the area of cavitation using machine learning image analysis. Open source software is used for image analysis. The image analysis presented is easy to replicate, does not require programming experience, and can be modified easily to suit the application of the user.

Cavitation microbubbles are imaged using a high-speed camera attached to a zoom lens. The experimental setup is explained, and image analysis is used to calculate the area of the cavitation. Image analysis is done using ImageJ.

التصوير والتدين الكمي لمنطقة الزبلات الدقيقة سريعة الحركة باستخدام كاميرا عالية السرعة وتحليل الصور

An experimental and image analysis technique is presented for imaging cavitation bubbles and calculating their area. The high-speed imaging experimental ...

Labeling of Extracellular Vesicles for Monitoring Migration and Uptake in Cartilage Explants

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their cellular source, such as platelet lysate (PL). When used as treatment, EVs are expected to enter the target cells and effect a response from these. In this research, PL-derived EVs have been studied as a cell-free treatment for osteoarthritis (OA). Thus, a method was set up to label EVs and test their uptake on cartilage explants. PL-derived EVs are labeled with the lipophilic dye PKH26, washed twice through a column, and then tested in an in vitro inflammation-driven&#160;OA model for 5 h after particle quantification by nanoparticle tracking analysis (NTA). Hourly, cartilage explants are fixed, paraffined, cut into 6 &#181;m sections to mount on slides, and observed under a confocal microscope. This allows verification of whether EVs enter the target cells (chondrocytes) during this period and analyze their direct effect.

Here, we present a protocol to label platelet lysate-derived extracellular vesicles to monitor their migration and uptake in cartilage explants used as a model for osteoarthritis.