<ol>
	<li>Tan, J. L., Tien, J., Pirone, D., Gray, D. S., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+lying+on+a+bed+of+microneedles:+An+approach+to+isolate+mechanical+force.">Cells lying on a bed of microneedles: An approach to isolate mechanical force.</a> Proc Nat Acad Sci USA. 100, 1484-1489 (2003).</li><li>Sniadecki, N. J., Chen, C. S., Wang, Y. L., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfabricated+Silicone+Elastomeric+Post+Arrays+for+Measuring+Traction+Forces+of+Adherent+Cells.">Microfabricated Silicone Elastomeric Post Arrays for Measuring Traction Forces of Adherent Cells.</a> Methods in Cell Biology - Cell Mechanics. 83, 313-328 (2007).</li><li>Lemmon, C. A., Sniadecki, N. J., Ruiz, S. A., Tan, J. T., Romer, L. H., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shear+Force+at+the+Cell-Matrix+Interface:+Enhanced+Analysis+For+Microfabricated+Post+Array+Detectors.">Shear Force at the Cell-Matrix Interface: Enhanced Analysis For Microfabricated Post Array Detectors.</a> Mechanics &amp; Chemistry of Biosystems. 2, 1-16 (2005).</li></ol>

microfabricated post-array-detectors (mpads): an approach to isolate mechanical forces

في هذا الفيديو ، وسوف نقدم نهجنا لقياس قوى الشد الخلوية باستخدام مجموعة microfabricated الوظائف. يتم إنشاؤها من خلال جر القوات الميوسين أكتين - التفاعلات وتلعب دورا هاما في علم وظائف الأعضاء لدينا. خلال التنمية ، فإنها تمكن الخلايا للانتقال من مكان واحد لآخر من أجل تشكيل الهياكل في وقت مبكر من الأنسجة. قوات الجر مساعدة في عمليات الشفاء. كانت ضرورية لإقفال السليم للجروح أو الهجرة والزحف من الكريات البيضاء من خلال جسمنا. يمكن لهذه القوى نفسها أن تكون ضارة على صحتنا في حالة ورم خبيث أو نمو الأوعية الدموية السرطانية نحو الورم. كانت الطريقة الأكثر شيوعا التي يمكن من خلالها دراسة الخلايا في المختبر لاستخدام الزجاج أو صحن البوليسترين. ومع ذلك ، فإن صلابة من ركائز يجعل من المستحيل فعليا على قياس قوى الجر الخلية ، وهناك طرق قليلة نسبيا لدراسة قوى الجر. وقد وضعت مختبرنا تقنية للتغلب على هذه القيود. يستند هذا الأسلوب على مجموعة العمودي للالكابولي مرنة ، وصلابة ومقياس حجم وهي ان هذه الخلايا الفردية موزعة على العديد من الكابولي وصرف لهم في هذه العملية. الركائز التي نستخدمها هي 3 ميكرومتر في القطر ، و 10 ميكرون طويل القامة ، ويتم تكوينها في مجموعة منتظمة مع تباعد 9 الوسط إلى مركز ميكرون. ولكن يمكن أن تكون هذه الأبعاد المادية المتنوعة بسهولة لاستيعاب مجموعة متنوعة من الدراسات. نبدأ مع رئيسي السيليكون ، ولكن يتم إجراء الوظائف النهائي من المطاط والسيليكون يسمى بولي (ميثيل siloxane) ، أو PDMS. يمكننا قياس الانحرافات تحت المجهر وحساب حجم واتجاه قوى الجر اللازمة لإنتاج الانحرافات الملاحظة. نحن ندعو هذه ركائز microfabricated بعد الصفيف كاشفات ، أو mPADs. هنا ، سوف نظهر لكم كيف نبتدع واستخدام mPADs لتقييم التحويرات من انقباض الخلوية.

Watch this Scientific Journal Video about Microfabricated Post-Array-Detectors mPADs: an Approach to Isolate Mechanical Forces at JoVE.com

Microfabricated Post-Array-Detectors mPADs: an Approach to Isolate Mechanical Forces

في هذا الفيديو ، ونحن لشرح كيفية تصنيع واستخدام أجهزة الكشف عن مجموعة microfabricated آخر (mPADs) لتقييم التحويرات من انقباض الخلوية.

Microfabricated بعد صفيف ، للكشف عن (mPADs) : نهج لعزل القوات الميكانيكية

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated ...

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Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces

12.4K Views.  University of Pennsylvania. في هذا الفيديو ، ونحن لشرح كيفية تصنيع واستخدام أجهزة الكشف عن مجموعة microfabricated آخر (mPADs) لتقييم التحويرات من انقباض الخلوية.

Video: Microfabricated Post-Array-Detectors mPADs: an Approach to Isolate Mechanical Forces

Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools

Using Micro-Electro-Mechanical Systems MEMS to Develop Diagnostic Tools

باستخدام مايكرو سيستمز الكهربائية والميكانيكية (ممس) لتطوير أدوات التشخيص

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

و السابقين البيضة الثقافة الأجنة النظام الدجاج مناسبة للتطبيقات التصوير والجراحة المجهرية

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

باستخدام SecM تسلسل توقيف كأداة لعزل البروتينية منضم ريبوسوم

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to assemble. Therefore, to identify these functional protein complexes, it is important to stabilize them in vivo before cell lysis and subsequent purification. Here we describe a method used to isolate large bona fide protein complexes from Drosophila embryos. This method is based on embryo permeabilization and stabilization of the complexes inside the embryos by in vivo crosslinking using a low concentration of formaldehyde, which can easily cross the cell membrane. Subsequently, the protein complex of interest is immunopurified followed by gel purification and analyzed by mass spectrometry. We illustrate this method using purification of a Tudor protein complex, which is essential for germline development. Tudor is a large protein, which contains multiple Tudor domains - small modules that interact with methylated arginines or lysines of target proteins. This method can be adapted for isolation of native protein complexes from different organisms and tissues.

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Multi-component protein complexes play crucial roles during cellular function and development. Here we describe a method used to isolate native protein complexes from Drosophila embryos after in vivo crosslinking followed by purification of the crosslinked complexes for subsequent structure-function analysis.

و في الجسم الحي يشابك النهج لعزل البروتين المجمعات من ذبابة الفاكهة الأجنة

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to ...

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

طريقة بديلة لإعداد نموذج مع انخفاض عدد الخلايا لتحليل تيم

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

تقييم لاستجابة ديسكويديوم ديكتيوستيليوم "تحفيز الميكانيكية الحادة"

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

مغناطيسية تنشيط الخلية فرز الاستراتيجيات الرامية إلى عزل وتنقية الزليلي المستمدة من سائل الخلايا الجذعية الوسيطة من طراز الأرنب

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

Protrusion Force Microscopy: A Method to Quantify Forces Developed by Cell Protrusions

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction forces have been well-characterized, but there is a lack of techniques allowing the measurement of the protrusion forces exerted by cells orthogonally to their substrate. We designed an experimental setup to measure the protrusion forces exerted by adherent cells on their substrate. Cells plated on a compliant Formvar sheet deform this substrate and the resulting topography is mapped by atomic force microscopy (AFM) at the nanometer scale. Force values are then extracted from an analysis of the deformation profile based on the geometry of the protrusive cellular structures. Hence, the forces exerted by the individual protruding units of a living cell can be measured over time. This technique will enable the study of force generation and its regulation in the many cellular processes involving protrusion. Here, we describe its application to measure the protrusive forces generated by podosomes formed by human macrophages.

Here, we detail the experimental techniques used to evaluate the protrusion forces that podosomes apply on a compliant film, from the preparation of the film to the automated analysis of topographical images.

مجهر القوة نتوء: أسلوب لتحديد حجم القوات المتقدمة بنتوءات الخلية

In numerous biological contexts, animal cells need to interact physically with their environment by developing mechanical forces. Among these, traction ...

A High Output Method to Isolate Cerebral Pericytes from Mouse

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood brain barrier formation and physiology is now demonstrated but its molecular basis remains largely unknown. As the pathophysiological role of cerebral pericytes in neurological disorders is intriguing and of great importance, the in vitro models are not only sufficiently appropriate but also able to incorporate different techniques for these studies. Several methods have been proposed as in vitro models for the extraction of cerebral pericytes, although an antibiotic-free protocol with high output is desirable. Most importantly, a method that has increased output per extraction reduces the usage of more animals.
Here, we propose a simple and efficient method for extracting cerebral pericytes with sufficiently high output. The mouse brain tissue homogenate is mixed with a BSA-dextran solution for the separation of the tissue debris and microvascular pellet. We propose a three-step separation followed by filtration to obtain a microvessel rich filtrate. With this method, the quantity of microvascular fragments obtained from 10 mice is sufficient to seed 9 wells (9.6 cm2 each) of a 6-well plate. Most interestingly with this protocol, the user can obtain 27 pericyte rich wells (9.6 cm2 each) in passage 2. The purity of the pericyte cultures are confirmed with the expression of classical pericyte markers: NG2, PDGFR-&#946; and CD146. This method demonstrates an efficient and feasible in vitro tool for physiological and pathophysiological studies on pericytes.

We present a protocol for the extraction of murine cerebral pericytes. Based on an antibiotic-free enrichment oriented pericyte extraction, this protocol is a valuable tool for in vitro studies providing high purity and high yield, thus decreasing the number of experimental animals used.

طريقه إخراج عاليه لعزل Pericytes الدماغي من الماوس

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood ...

A Finite Element Approach for Locating the Center of Resistance of Maxillary Teeth

The center of resistance (CRES) is regarded as the fundamental reference point for predictable tooth movement. The methods used to estimate the CRES of teeth range from traditional radiographic and physical measurements to in vitro analysis on models or cadaver specimens. Techniques involving finite element analysis of high-dose micro-CT scans of models and single teeth have shown a lot of promise, but little has been done with newer, low-dose, and low resolution cone beam computed tomography (CBCT) images. Also, the CRES for only a few select teeth (i.e., maxillary central incisor, canine, and first molar) have been described; the rest have been largely ignored. There is also a need to describe the methodology of determining the CRES in detail, so that it becomes easy to replicate and build upon.
This study used routine CBCT patient images for developing tools and a workflow to obtain finite element models for locating the CRES of maxillary teeth. The CBCT volume images were manipulated to extract three-dimensional (3D) biological structures relevant in determining the CRES of the maxillary teeth by segmentation. The segmented objects were cleaned and converted into a virtual mesh made up tetrahedral (tet4) triangles having a maximum edge length of 1 mm with 3matic software. The models were further converted into a solid volumetric mesh of tetrahedrons with a maximum edge length of 1 mm for use in finite element analysis. The engineering software, Abaqus, was used to preprocess the models to create an assembly and set material properties, interaction conditions, boundary conditions, and load applications. The loads, when analyzed, simulated the stresses and strains on the system, aiding in locating the CRES. This study is the first step in accurate prediction of tooth movement.

This study outlines the necessary tools for utilizing low-dose three-dimensional cone beam-based patient images of the maxilla and maxillary teeth to obtain finite element models. These patient models are then used to accurately locate the CRES of all the maxillary teeth.

نهج عنصر محدود لتحديد موقع مركز مقاومة الأسنان الفكية

The center of resistance (CRES) is regarded as the fundamental reference point for predictable tooth movement. The methods used to estimate the CRES of ...