نود أن نشكر مختبر Blelloch عن التعليق على النص. وأيد هذا العمل من جانب صناديق لRB من المعاهد الوطنية للصحة (K08 NS48118 وNS057221 R01) ، معهد كاليفورنيا للطب التجديدي (CIRM) (المنحة البذور RS1 - 00161 ، ونيو جائزة كلية RN2 - 00906) ، وصندوق بيو الخيرية وزير الخارجية من Wissenschaftlich Urologische غزلشافت فولت.

<ol>
	<li>Mitchell, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+microRNAs+as+stable+blood-based+markers+for+cancer+detection.">Circulating microRNAs as stable blood-based markers for cancer detection.</a> Proc Natl Acad Sci U S A. 105, 10513-10518 (2008).</li><li>Tang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=220-plex+microRNA+expression+profile+of+a+single+cell.">220-plex microRNA expression profile of a single cell.</a> Nat Protoc. 1, 1154-1159 (2006).</li><li>Gertsenstein, M., Vintersten, K., Behringer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the Mouse Embryo: A Laboratory Manual. 3, 644-645 (2003).</li><li>Suh, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+function+is+globally+suppressed+in+mouse+oocytes+and+early+embryos.">MicroRNA function is globally suppressed in mouse oocytes and early embryos.</a> Curr Biol. 20, 271-277 (2010).</li><li>Babiarz, J. E., Ruby, J. G., Wang, Y., Bartel, D. P., Blelloch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+ES+cells+express+endogenous+shRNAs,+siRNAs,+and+other+Microprocessor-independent,+Dicer-dependent+small+RNAs.">Mouse ES cells express endogenous shRNAs, siRNAs, and other Microprocessor-independent, Dicer-dependent small RNAs.</a> Genes Dev. 22, 2773-2785 (2008).</li><li>Babiarz, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+RNAs:+their+biogenesis,+regulation+and+function+in+embryonic+stem+cells.">Small RNAs: their biogenesis, regulation and function in embryonic stem cells.</a> StemBook. , (2009).</li><li>Friedman, R. C., Farh, K. K., Burge, C. B., Bartel, 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=Most+mammalian+mRNAs+are+conserved+targets+of+microRNAs.">Most mammalian mRNAs are conserved targets of microRNAs.</a> Genome Res. 19, 92-105 (2008).</li><li>Wang, Y., Medvid, R., Melton, C., Jaenisch, R., Blelloch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DGCR8+is+essential+for+microRNA+biogenesis+and+silencing+of+embryonic+stem+cell+self-renewal.">DGCR8 is essential for microRNA biogenesis and silencing of embryonic stem cell self-renewal.</a> Nat Genet. 39, 380-385 (2007).</li><li>Kanellopoulou, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dicer-deficient+mouse+embryonic+stem+cells+are+defective+in+differentiation+and+centromeric+silencing.">Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing.</a> Genes Dev. 19, 489-501 (2005).</li><li>Thomson, J. M., Parker, J., Perou, C. M., Hammond, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+custom+microarray+platform+for+analysis+of+microRNA+gene+expression.">A custom microarray platform for analysis of microRNA gene expression.</a> Nat Methods. 1, 47-53 (2004).</li><li>Moltzahn, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+based+multiplex+qRT-PCR+identifies+diagnostic+and+prognostic+microRNA+signatures+in+sera+of+prostate+cancer+patients.">Microfluidic based multiplex qRT-PCR identifies diagnostic and prognostic microRNA signatures in sera of prostate cancer patients.</a> Cancer Res. 71, 550-560 (2011).</li><li>Mestdagh, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+and+universal+method+for+microRNA+RT-qPCR+data+normalization.">A novel and universal method for microRNA RT-qPCR data normalization.</a> Genome Biol. 10, R64-R64 (2009).</li></ol>

آلان مير هو موظف في شركة Fluidigm. خلاف ذلك ، ليست لدينا مصالح مالية في الكشف عنها.

miRNAs قصيرة (18-24 النيوكليوتيدات) ، غير الترميز RNAs وميرنا ، التي تنظم التعبير الجيني في مرحلة ما بعد transcriptionally من قبل كل من الرنا الرسول زعزعة الاستقرار (مرنا) ، وتحول دون ترجمتها. 6 وحقيقة أن ما لا يقل عن ثلث الجينات البشرية تحتوي على الحفظ ملزمة مواقع في 3&#39;UTR والتفاعل واضحا من miRNAs مع الجينات للانتشار ، وموت الخلايا المبرمج تعدد القدرات تقترح مساهمة حاسمة في اتخاذ القرارات مصير الخلية ، الأنسجة والتوازن أمراض مثل السرطان. microRNA التعبير 6،7 دقيقة لذلك هي الملامح العريضة الفائدة. لاختبار متعددة نشرت qRT - PCR التنميط التعبير بروتوكول ميرنا 2 استخدمنا الصغيرة RNA MESC أنها مقصرة ضوابط السلبية. DGCR8 -- / -- تعاني من نقص خلايا جميع microRNAs الكنسي ، في حين أن المقامر -- / -- نقص خلايا micoRNAs الكنسي على حد سواء وعدم canoncial - 8،9 مقارنة مستويات في نوع البرية MES إلى DGCR8 -- / -- الخلايا ، وكشف لنا عدم وجود دقة وأعرب عن عدة لم يتم اكتشاف 10 microRNAs في الخلايا البرية من نوع 11. وأظهرت بعض حتى انخفاض مستويات التعبير النسبي للخلايا خروج المغلوب (الشكل 3A). افترضنا أنه قد يكون السبب في عدم مراعاة الدقة من قبل التمهيدي ضخمة تحمل أكثر من خطوتين متتاليتين متعددة (RT PCR وتمهيدي) قبل الكمي - RT singleplex. ومع ذلك ، متعددة قبل التضخيم ضروري لتحسين قوة الإشارة ، وخصوصا عندما يقتصر تركيز بدءا من الحمض النووي الريبي. بعيدا عن طريق تنقية قبل PCR المنتجات من الاشعال عن طريق الانتقاء في حجم المواد الهلامية بولي أكريلاميد الأصلي (الشكل 2) ، فإننا يمكن أن يبرهن على وجود تحسن كبير في الدقة عن طريق الكشف عن مزيد من microRNAs وفقدان اشارات ايجابية كاذبة في كل Dgcr8 -- / -- والمقامر -- / -- خلفيات (أرقام 3a و 3B) بالإضافة إلى تعديل qRT - PCR النهج سمح لتصنيف المناسبة النادرة Dgcr8 الرناوات المستقل / المقامر صغيرة تعتمد على (الشكل 3B) 11. بالتالي خطوة إضافية لتنقية الاشعال المفرط بعيدا عن المنتج قبل PCR هو مفيد بشكل واضح ، خاصة عند استخدام مجموعات كبيرة التمهيدي متعددة لكل عينة. يتم تحديد العدد الأمثل للدورات السابقة للتضخيم وفقا لتركيزات الحمض النووي الريبي الإدخال. وينبغي أن يسترشد على التوازن الذي لا يخضع لأو overamplify بواسطة تفاصيل تجربة محددة. مع أكثر من ألف microRNAs المعروفة ، واستخدام معيار 384 لوحات جيدا قد لا تكون الأمثل لالتنميط microRNA واسعة النطاق ، وخصوصا عندما تقارن عينات متعددة. Fluidigm مؤسسة التمويل الدولية في صفيف حيوية تمكن ، مع انخفاض كبير في عدد الخطوات اللازمة pipetting والكيمياء ، واختبار ما يصل الى 96 عينات الفرد من 96 microRNAs مختلفة في تجربة واحدة (9216 ردود فعل) على نطاق واسع نانولتر (6.7 NL). لكل تشغيل البرنامج يوفر تحليل المنحنيات التضخيم ، وخرائط الحرارة مرمزة والقيم عتبة دورة (CT). في وقت واحد على نطاق واسع التنميط يقلل الفروق التجريبية ، ويتيح التعبير يعني التطبيع قيمة ، والذي ينفذ خارج استراتيجيات التطبيع الأخرى التي تجعل من استخدام الضوابط RNA الصغيرة (12). بالمقارنة مع 384 منصات متوفرة تجاريا جيدا مع تحقيقات TaqMan قبل تعيينه ، فإن الجمع بين منصة عالية الإنتاجية التنميط مع مجموعات التمهيدي مصنوعة خصيصا يوفر مرونة عالية التجريبية. والأمثل متعددة qRT - PCR النهج في تركيبة مع منصة صفيف الحيوي يسمح بنجاح الشاشة في وقت واحد منا أن 48 مريض السرطان الأمصال البروستاتا لإحداث تغييرات في مستويات من 384 ميرنا (الشكل 4) وتطبيع البيانات دون زيادة كبيرة في التحكم (أي الاصطناعية microRNAs). 11 على الرغم من الزيادة من الخطوات من العينات تمهيدا لنتائج التنميط ، والنهج صفها هو الوقت والتكلفة الفعالة أسلوب إنتاجية عالية إلى ملف مجموعات عينة كبيرة لمستويات التعبير ميرنا ، حتى مع محدودية المواد البداية.

high throughput microrna profiling: optimized multiplex qrt-pcr at nanoliter scale on the fluidigm dynamic arraytm ifcs

وقد أدى تدخل واسع من العمليات الحرجة في miRNAs الكامنة التنمية ، والأنسجة homoeostasis المرض إلى اهتمام المتزايد بين المجتمعات والبحوث الدوائية. لدراسة miRNAs ، فمن الضروري أن القياس الكمي لمستويات microRNA دقيقة وقوية. بمقارنة البرية من نوع خلايا الحمض النووي الريبي للمشاريع الصغيرة الجذعية الجنينية ناقصة (مسك) ، وكشف لنا عدم وجود دقة ومتانة في السابق نشر تقنيات متعددة qRT - PCR. هنا ، نحن تصف أسلوب الأمثل ، بما في ذلك تنقية بعيدا الاشعال المفرط من الخطوات السابقة قبل الكشف المتعدد singleplex الوقت الحقيقي ، مما يزيد بشكل كبير من دقة ومتانة الأسلوب. علاوة على ذلك ، يمكننا أن نفسر كيف يؤدون هذه التقنية على رقاقة ميكروفلويديك في أحجام نانولتر يقلل كثيرا من تكاليف كاشف وسمح الوقت فعالة ميرنا إنتاجية عالية التنميط التعبير.

Watch this Scientific Journal Video about High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs at JoVE.com

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

نحن هنا تصف عكس متعددة الأمثل المنتسخة PCR الكمي (qRT - PCR) بروتوكول بالاشتراك مع منصة ميكروفلويديك من حيث التكلفة والوقت أداة فعالة الفرز الفائق الإنتاجية للmicroRNA (ميرنا) مستويات التعبير ، وخاصة عند العمل مع كميات محدودة من العينة.

ارتفاع جانبي MicroRNA المناولة : ملتيبليكس محسن qRT - PCR على مقياس نانولتر في المحفل Fluidigm ArrayTM الحيوي

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

high-throughput-microrna-profiling-optimized-multiplex-qrt-pcr-at

Research

JoVE Journal

Bioengineering

20.4K Views.  University of California San Francisco. نحن هنا تصف عكس متعددة الأمثل المنتسخة PCR الكمي (qRT - PCR) بروتوكول بالاشتراك مع منصة ميكروفلويديك من حيث التكلفة والوقت أداة فعالة الفرز الفائق الإنتاجية للmicroRNA (ميرنا) مستويات التعبير ، وخاصة عند العمل مع كميات محدودة من العينة.

Video: High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates. It is ideal for medium- to high-throughput screens in academic settings. It has an integrated fluid transfer module equipped with a multi-channel pipetter and the machine reads one column at a time to monitor fluorescence changes of a variety of fluorescent reagents. For example, FlexStation 3 has been used to study the function of Ca2+-permeable ion channels and G-protein coupled receptors by measuring the changes of intracellular free Ca2+ levels. Transient receptor potential (TRP) channels are a large family of nonselective cation channels that play important roles in many physiological and pathophysiological functions. Most of the TRP channels are calcium permeable and induce calcium influx upon activation. In this video, we demonstrate the application of FlexStation 3 to study the pharmacological profile of the TRPA1 channel, a molecular sensor for numerous noxious stimuli. HEK293 cells transiently or stably expressing human TRPA1 channels, grown in 96-well plates, are loaded with a Ca2+-sensitive fluorescent dye, Fluo-4, and real-time fluorescence changes in these cells are measured before and during the application of a TRPA1 agonist using the FLEX mode of the FlexStation 3. The effect of a putative TRPA1 antagonist was also examined. Data are transferred from the SoftMax Pro software to construct concentration-response relationships of TRPA1 activators and inhibitors.

This video provides a detailed protocol for studying the pharmacological profile of human TRPA1 channels using FlexStation 3. The protocol covers details of cell preparation, dye loading and operation of the microplate reader, FlexStation 3.

الخلية المستندة إلى الفحص الكالسيوم لمتوسطة إلى فحص وظائف إنتاجية عالية باستخدام قناة الحزب FlexStation 3

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates.  It is ...

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

المبيضات البيض بيوفيلم رقاقة (كاليفورنيا BChip) لالفرز الفائق الإنتاجية مضاد للمخدرات

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

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 ...

Synthetic Spider Silk Production on a Laboratory Scale

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation biomaterials with high performance properties. The development of these new structural materials must be rapid, cost-efficient and involve processing methodologies and products that are environmentally friendly and sustainable. Spiders spin a multitude of different fiber types with diverse mechanical properties, offering a rich source of next generation engineering materials for biomimicry that rival the best manmade and natural materials. Since the collection of large quantities of natural spider silk is impractical, synthetic silk production has the ability to provide scientists with access to an unlimited supply of threads. Therefore, if the spinning process can be streamlined and perfected, artificial spider fibers have the potential use for a broad range of applications ranging from body armor, surgical sutures, ropes and cables, tires, strings for musical instruments, and composites for aviation and aerospace technology. In order to advance the synthetic silk production process and to yield fibers that display low variance in their material properties from spin to spin, we developed a wet-spinning protocol that integrates expression of recombinant spider silk proteins in bacteria, purification and concentration of the proteins, followed by fiber extrusion and a mechanical post-spin treatment. This is the first visual representation that reveals a step-by-step process to spin and analyze artificial silk fibers on a laboratory scale. It also provides details to minimize the introduction of variability among fibers spun from the same spinning dope. Collectively, these methods will propel the process of artificial silk production, leading to higher quality fibers that surpass natural spider silks.

Despite the outstanding mechanical and biochemical properties of spider silks, this material cannot be harvested in large quantities by conventional means. Here we describe an efficient strategy to spin artificial spider silk fibers, which is an important process for investigators studying spider silk production and their use as next-generation biomaterials.

الاصطناعية إنتاج حرير العنكبوت على نطاق المختبر

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation ...

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high packaging capacity (up to 35 kb), low immunogenicity, and low toxicity. However, for many laboratories the use of HCAdV is hampered by the complicated procedure for vector genome construction and virus production. Here, a detailed protocol for efficient cloning and production of HCAdV based on the plasmid pAdFTC containing the HCAdV genome is described. The construction of HCAdV genomes is based on a cloning vector system utilizing homing endonucleases (I-CeuI and PI-SceI). Any gene of interest of up to 14 kb can be subcloned into the shuttle vector pHM5, which contains a multiple cloning site flanked by I-CeuI and PI-SceI. After I-CeuI and PI-SceI-mediated release of the transgene from the shuttle vector the transgene can be inserted into the HCAdV cloning vector pAdFTC. Because of the large size of the pAdFTC plasmid and the long recognition sites of the used enzymes associated with strong DNA binding, careful handling of the cloning fragments is needed. For virus production, the HCAdV genome is released by NotI digest and transfected into a HEK293 based producer cell line stably expressing Cre recombinase. To provide all adenoviral genes for adenovirus amplification, co-infection with a helper virus containing a packing signal flanked by loxP sites is required. Pre-amplification of the vector is performed in producer cells grown on surfaces and large-scale amplification of the vector is conducted in spinner flasks with producer cells grown in suspension. For virus purification, two ultracentrifugation steps based on cesium chloride gradients are performed followed by dialysis. Here tips, tricks, and shortcuts developed over the past years working with this HCAdV vector system are presented.

A protocol for generation of high-capacity adenoviral vectors lacking all viral coding sequences is presented. Cloning of transgenes contained in the vector genome is based on homing endonucleases. Virus amplification in producer cells grown as adherent cells and in suspension relies on a helper virus providing viral genes in trans.

الاستنساخ واسع النطاق إنتاج ذات قدرة عالية ناقلات الفيروسة الغدانية استنادا إلى نوع اتش 5 الإنسان

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high ...

Easy and Accurate Mechano-profiling on Micropost Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate surroundings. Particularly, cell-substrate interactions are the main interest. Today micropost arrays are a well-characterized and established method with a broad range of applications that have been published over the last decade. However, there seems to be a reservation among biologists to adapt the technique due to the lengthy and challenging process of micropost manufacture along with the lack of easily approachable software for analyzing images of cells interacting with microposts. 
The force read-out from microposts is surprisingly easy. A micropost acts like a spring with the cell ideally attached at its tip. Depending on size a cell applies force from its cytoskeleton through one or multiple focal adhesion points to the micropost, thus deflecting the micropost. The amount of deflection correlates directly to the applied force in direction and in magnitude. The number of microposts covered by a cell and the post deflection patterns are characteristic and allow determination of values like force per post and many biologically relevant parameters that allow &#8220;mechano-profiling&#8221; of cell phenotypes.
A convenient method for mechano-profiling is described here combining the first generation of ready-to-use commercially available microposts with an in-house developed software package that is now accessible to all researchers. As a demonstration of typical application, single images of bone cancer cells were taken in bright-field microscopy for mechano-profiling of cell line models of metastasis. This combination of commercial traction force sensors and open source software for analysis allows for the first time a rapid implementation of the micropost array technique into routine lab work done by non-expert users. Furthermore, a robust and streamlined analysis process enables a user to analyze a large number of micropost images in a highly time-efficient manner.

Described are protocols for quantifying mechanical interactions between adherent cells and microstructured substrates. These interactions are closely linked to essential cell behaviors including migration, proliferation, differentiation, and apoptosis. The protocols present an open-source image analysis software called MechProfiler, which enables determination of involved forces for each micropost.

سهلة ودقيقة والميكانيكية التنميط على Micropost صالحة

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate ...

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

باستخدام Tomoauto: بروتوكول لإنتاجية عالية الآلي البرد الإلكترون التصوير المقطعي

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

وإنتاجية عالية منصة ميكروأري الخليوي لتحليل مترابط من تمايز الخلايا وقوات الجر

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

Power Input Measurements in Stirred Bioreactors at Laboratory Scale

The power input in stirred bioreactors is an important scaling-up parameter and can be measured through the torque that acts on the impeller shaft during rotation. However, the experimental determination of the power input in small-scale vessels is still challenging due to relatively high friction losses inside typically used bushings, bearings and/or shaft seals and the accuracy of commercially available torque meters. Thus, only limited data for small-scale bioreactors, in particular single-use systems, is available in the literature, making comparisons among different single-use systems and their conventional counterparts difficult.
This manuscript provides a protocol on how to measure power inputs in benchtop scale bioreactors over a wide range of turbulence conditions, which can be described by the dimensionless Reynolds number (Re). The aforementioned friction losses are effectively reduced by the use of an air bearing. The procedure on how to set up, conduct and evaluate a torque-based power input measurement, with special focus on cell culture typical agitation conditions with low to moderate turbulence (100 &#60; Re &#60; 2&#183;104), is described in detail. The power input of several multi-use and single-use bioreactors is provided by the dimensionless power number (also called Newton number, P0), which is determined to be in the range of P0 &#8776; 0.3 and P0 &#8776; 4.5 for the maximum Reynolds numbers in the different bioreactors.

The power input in stirred bioreactors can be measured through the torque that acts on the impeller shaft during rotation. This manuscript describes how an air bearing can be used to effectively reduce friction losses observed in mechanical seals and improve the accuracy of power input measurements in small-scale vessels.

قياسات الطاقة الإدخال في المفاعلات الحيوية المقلبة في المختبرات

The power input in stirred bioreactors is an important scaling-up parameter and can be measured through the torque that acts on the impeller shaft during ...

Environmental Dynamic Mechanical Analysis to Predict the Softening Behavior of Neural Implants

When using dynamically softening substrates for neural implants, it is important to have a reliable in vitro method to characterize the softening behavior of these materials. In the past, it has not been possible to satisfactorily measure the softening of thin films under conditions mimicking body environment without substantial effort. This publication presents a new and simple method that allows dynamic mechanical analysis (DMA) of polymers in solutions, such as phosphate buffered saline (PBS), at relevant temperatures. The use of environmental DMA allows measurement of the softening effects of polymers due to plasticization in various media and temperatures, which therefore allows a prediction of the materials behavior under in vivo conditions.

To allow reliable predictions of the softening of polymeric substrates for neural implants in an in vivo environment, it is important to have a reliable in vitro method. Here, the use of dynamic mechanical analysis in phosphate buffered saline at body temperature is presented.

تحليل الميكانيكية الحيوية البيئية للتنبؤ بسلوك تليين يزرع العصبية

When using dynamically softening substrates for neural implants, it is important to have a reliable in vitro method to characterize the softening behavior ...