وأيد هذه الدراسة من قبل المعاهد الوطنية للصحة منح R01HL091020 ، R01HL093723 ، R01AI077343 وR01GM096187.

<ol>
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الإعلان عن أي تضارب في المصالح.

لاستخدام بنجاح وتيرة التصاق مقايسة micropipette ينبغي للمرء أن ينظر في اتخاذ خطوات حاسمة عدة. أولا ، تأكد من أن يسجل التفاعل محددة لنظام مستقبلات يجند من الفائدة. قياسات التحكم غير محدد (راجع الشكل رقم 3 ، 4) ضمان النوعية. من الناحية المثالية ، ينبغي أن تكون احتمالات التصاق غ?...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><th> اسم الكاشف </th><th> شركة </th><th> كتالوج # </th><th> تعليقات </th></tr><tr><td> 10X PBS </td><td> BioWhittaker </td><td> 17 - 517Q </td><td> المخفف بالماء ل1X منزوع الأيونات قبل استخدامها </td></tr><tr><td> Vacutainer EDTA </td><td> دينار بحريني </td><td> 366643 </td><td> كرات الدم الحمراء العزلة </td></tr><tr><td> 10ML PK100 </td><td></td><td></td><td></td></tr><tr><td> Histopaque 1077 </td><td> سيغما الدريخ </td><td> 10771 </td><td> كرات الدم الحمراء العزلة </td></tr><tr><td> الأدينين </td><td> سيغما الدريخ </td><td> A2786 </td><td> EAS - 45 إعداد </td></tr><tr><td> مد الجلوكوز (سكر العنب) </td><td> سيغما الدريخ </td><td> G7528 </td><td> EAS - 45 إعداد </td></tr><tr><td> D - المانيتول </td><td> سيغما الدريخ </td><td> 6360 </td><td> EAS - 45 إعداد </td></tr><tr><td> كلوريد الصوديوم (كلوريد الصوديوم) </td><td> سيغما الدريخ </td><td> S7653 </td><td> EAS - 45 إعداد </td></tr><tr><td> فوسفات الصوديوم ، ثنائي القاعدة (نا س &amp; #2082 ؛ هبو ₄) </td><td> فيشر العلمية </td><td> S374 </td><td> EAS - 45 إعداد </td></tr><tr><td> L - الجلوتامين </td><td> سيغما الدريخ </td><td> G5763 </td><td> EAS - 45 إعداد </td></tr><tr><td> البيوتين - X - NHS </td><td> Calbiochem </td><td> 203188 </td><td> كرات الدم الحمراء biotinylation </td></tr><tr><td> ثنائي ميثيل الفورماميد (DMF) </td><td> الحرارية العلمية </td><td> 20673 </td><td> كرات الدم الحمراء biotinylation </td></tr><tr><td> بورات العازلة (0.1M) </td><td> الإلكترون المجهري العلوم </td><td> 11455-90 </td><td> كرات الدم الحمراء biotinylation </td></tr><tr><td> Streptavidin </td><td> الحرارية العلمية </td><td> 21125 </td><td> يجند functionalizing </td></tr><tr><td> BSA </td><td> سيغما الدريخ </td><td> A0336 </td><td> يجند functionalizing </td></tr><tr><td> Quantibrite الخرز PE </td><td> العلوم البيولوجية دينار بحريني </td><td> 340495 </td><td> كثافة الكمي </td></tr><tr><td> تدفق عداد الكريات </td><td> نظم دينار بحريني Immunocytometry </td><td> دينار بحريني LSR الثاني </td><td> كثافة تشيوانtification </td></tr><tr><td> الأنبوبة الشعرية 0.7 - 1.0mm × 30 &quot; </td><td> كيمبل شركة زجاج </td><td> 46485-1 </td><td> سحب Micropipette </td></tr><tr><td> الزيوت المعدنية </td><td> فيشر العلمية </td><td> BP2629 - 1 </td><td> غرفة التجميع </td></tr><tr><td> مجهر غطاء زجاج </td><td> فيشر العلمية </td><td> 12-544 - G </td><td> غرفة التجميع </td></tr><tr><td> PE - α الإنسان CD11a استنساخ مرحبا 111 </td><td> eBioscience </td><td> 12-0119-71 </td><td> كاشف للFig.1 </td></tr><tr><td> PE مكافحة CD54 الإنسان </td><td> eBioscience </td><td> 12-0549 </td><td> كاشف للFig.1 </td></tr><tr><td> الماوس IgG1 Isotype التحكم PE </td><td> eBioscience </td><td> 12-4714 </td><td> كاشف للFig.1 </td></tr><tr><td> الهيدروليكية micromanipulator </td><td> Narishige </td><td> MO - 303 </td><td> Micropipette النظام </td></tr><tr><td> مناور الميكانيكية </td><td> نيوبورت </td><td> 461 م ، XYZ ، SM - 13 ، DM - 13 </td><td> Micropipette النظام </td></tr><tr><td> كهرضغطية مترجم </td><td> Physik الرفيعة Instrumente </td><td> P - 840 </td><td> Micropipette النظام </td></tr><tr><td> ابفيو (LabVIEW) </td><td> الصكوك الوطنية </td><td> الإصدار 8.6 </td><td> Micropipette النظام </td></tr><tr><td> دق متنها </td><td> الصكوك الوطنية </td><td> USB - 6008 </td><td> Micropipette النظام </td></tr><tr><td> الجدول البصرية </td><td> نظم حركية </td><td> 5200 سلسلة </td><td> Micropipette النظام </td></tr></tbody></table>

adhesion frequency assay for in situ kinetics analysis of cross-junctional molecular interactions at the cell-cell interface

تم تطوير مقايسة التصاق micropipette في عام 1998 لقياس ثنائي الأبعاد (2D) مستقبلات يجند حركية ملزمة 1. في مقايسة يستخدم الإنسان خلايا الدم الحمراء (RBC) وأجهزة الاستشعار والتصاق الخلية عرض لواحدة من الجزيئات المتفاعلة. انها توظف المجهرية لجلب RBC في اتصال مع خلية أخرى يعبر عن جزيء التفاعل مع غيرها من المنطقة التي تسيطر على وجه التحديد والوقت لتمكين تشكيل السندات. تم الكشف عن حالة التصاق والاستطالة على سحب RBC الخلايا باستثناء اثنين. من خلال التحكم في كثافة يغاندس ثبتوا على سطح خلايا الدم الحمراء ، يتم الاحتفاظ احتمال الالتصاق في منتصف المدى بين 0 و 1. ويقدر احتمال التصاق من وتيرة الأحداث التصاق في سلسلة من دورات متكررة الاتصال بين خليتين لوقت الاتصال معين. متفاوتة في وقت الاتصال يولد منحنى ملزم. تركيب نموذج احتمالي لمستقبلات يجند رد فعل حركية 1 إلى الربطمنحنى عائدات تقارب معدل 2D وقبالة. وقد تم التحقق من صحة الفحص باستخدام تفاعلات مستقبلات Fcγ مع نادي مفتش 1-6 و selectins مع يغاندس glycoconjugate 6-9 ، لل integrins مع يغاندس 10-13 ، homotypical كادهيرين ملزمة 14 ، تي مستقبلات الخلية وcoreceptor مع المجمعات الكبرى التوافق النسيجي الببتيد - 15 -- 19. وقد استخدمت هذه الطريقة لقياس أنظمة حركية 2D عوامل البيولوجية والفيزيائية ، مثل الغشاء microtopology 5 ، 2 مرساة الغشاء ، والتوجه الجزيئية وطول 6 ، وتصلب الناقل 9 ، 20 انحناء ، وقوة اعتداءات 20 ، فضلا عن العوامل البيوكيميائية ، مثل جهري من الهيكل الخلوي والغشاء المكروية حيث تتفاعل الجزيئات الموجودة وتنظيم سطح هذه الجزيئات 15،17،19. الأسلوب كما استخدمت رس دراسة الربط المتزامن لأنواع مستقبلات يجند 3،4 المزدوج ، والتفاعلات trimolecular 19 باستخدام نموذج تعديل 21. والميزة الرئيسية لهذه الطريقة أنها تسمح دراسة المستقبلات في غشاء بيئتها الأصلية. ويمكن أن تكون النتائج مختلفة جدا عن تلك التي تم الحصول عليها باستخدام مستقبلات المنقى 17. كما يسمح دراسة التفاعلات مستقبلات يجند في فترة زمنية الفرعي الثاني للقرار الزمنية إلى ما وراء الأساليب التقليدية البيوكيميائية. لتوضيح طريقة التصاق micropipette التردد ، وتبين لنا قياس حركية جزيء التصاق بين الخلايا 1 (ICAM - 1) على كرات الدم الحمراء functionalized ملزمة لإنتغرين β α L 2 على العدلات مع مثنوي E - selectin في الحل لتنشيط β α L 2.

Watch this Scientific Journal Video about Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface at JoVE.com

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

يوصف لفحص لقياس التردد التصاق حركية التفاعل مستقبلات يجند عندما ترسو على حد سواء الجزيئات على أسطح الخلايا المتفاعلة. ويتمثل هذا الاختبار القائم على ميكانيكيا باستخدام micropipette مضغوطة الإنسان خلايا الدم الحمراء وأجهزة الاستشعار والتصاق αLβ2 إنتغرين وبين الخلايا جزيء التصاق - 1 والتفاعل ومستقبلات يغاندس.

التصاق الفحص عن التردد في الموقع تحليل حركية عبر صلي التفاعلات الجزيئية في خلية واجهة سيل

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

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Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

14.9K Views.  Georgia Institute of Technology. يوصف لفحص لقياس التردد التصاق حركية التفاعل مستقبلات يجند عندما ترسو على حد سواء الجزيئات على أسطح الخلايا المتفاعلة. ويتمثل هذا الاختبار القائم على ميكانيكيا باستخدام micropipette مضغوطة الإنسان خلايا الدم الحمراء وأجهزة الاستشعار والتصاق αLβ2 إنتغرين وبين الخلاي?...

Video: Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

ذات دقة عالية الألياف الضوئية للMicroendoscopy في الموقع الخلوية التصوير

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

Bridging the Bio-Electronic Interface with Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and higher throughput. The incorporation of biological components onto biological microelectromechanical systems (bioMEMS) has shown great potential for achieving these goals. Microfabricated electronic chips allow for micrometer-scale features as well as an electrical connection for sensing and actuation. Functional biological components give the system the capacity for specific detection of analytes, enzymatic functions, and whole-cell capabilities. Standard microfabrication processes and bio-analytical techniques have been successfully utilized for decades in the computer and biological industries, respectively. Their combination and interfacing in a lab-on-a-chip environment, however, brings forth new challenges. There is a call for techniques that can build an interface between the electrode and biological component that is mild and is easy to fabricate and pattern.
Biofabrication, described here, is one such approach that has shown great promise for its easy-to-assemble incorporation of biological components with versatility in the on-chip functions that are enabled. Biofabrication uses biological materials and biological mechanisms (self-assembly, enzymatic assembly) for bottom-up hierarchical assembly. While our labs have demonstrated these concepts in many formats 1,2,3, here we demonstrate the assembly process based on electrodeposition followed by multiple applications of signal-based interactions. The assembly process consists of the electrodeposition of biocompatible stimuli-responsive polymer films on electrodes and their subsequent functionalization with biological components such as DNA, enzymes, or live cells 4,5. Electrodeposition takes advantage of the pH gradient created at the surface of a biased electrode from the electrolysis of water 6,7,. Chitosan and alginate are stimuli-responsive biological polymers that can be triggered to self-assemble into hydrogel films in response to imposed electrical signals 8. The thickness of these hydrogels is determined by the extent to which the pH gradient extends from the electrode. This can be modified using varying current densities and deposition times 6,7. This protocol will describe how chitosan films are deposited and functionalized by covalently attaching biological components to the abundant primary amine groups present on the film through either enzymatic or electrochemical methods 9,10. Alginate films and their entrapment of live cells will also be addressed 11. Finally, the utility of biofabrication is demonstrated through examples of signal-based interaction, including chemical-to-electrical, cell-to-cell, and also enzyme-to-cell signal transmission. 
Both the electrodeposition and functionalization can be performed under near-physiological conditions without the need for reagents and thus spare labile biological components from harsh conditions. Additionally, both chitosan and alginate have long been used for biologically-relevant purposes 12,13. Overall, biofabrication, a rapid technique that can be simply performed on a benchtop, can be used for creating micron scale patterns of functional biological components on electrodes and can be used for a variety of lab-on-a-chip applications.

This article describes a biofabrication approach: deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications.

سد واجهة الحيوية الالكترونية مع Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

تحديد معدل النقل من الاكسيوبيوتك والمواد النانوية عبر المشيمة باستخدام<em&gt; خارج الحي</em&gt; الإنسان المشيمة الإرواء الموديل

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or erosion. Amelogenin has been proven to be a critical protein for controlling the organized growth of apatite crystals. In this paper, we present a detailed protocol for superficial enamel reconstruction by using a novel amelogenin-chitosan hydrogel. Compared to other conventional treatments, such as topical fluoride and mouthwash, this method not only has the potential to prevent the development of dental caries but also promotes significant and durable enamel restoration. The organized enamel-like microstructure regulated by amelogenin assemblies can significantly improve the mechanical properties of etched enamel, while the dense enamel-restoration interface formed by an in situ regrowth of apatite crystals can improve the effectiveness and durability of restorations. Furthermore, chitosan hydrogel is easy to use and can suppress bacterial infection, which is the major risk factor for the occurrence of dental caries. Therefore, this biocompatible and biodegradable amelogenin-chitosan hydrogel shows promise as a biomaterial for the prevention, restoration, and treatment of defective enamel.

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

In this article, we describe a protocol for fabricating an amelogenin-chitosan hydrogel for superficial enamel reconstruction. Organized in situ growth of apatite crystals in the hydrogel formed a dense enamel-restoration interface, which will improve the effectiveness and durability of restorations.

تطوير أميلوجينين-الشيتوزان هيدروجيل ل<em&gt; في المختبر</em&gt; إعادة نمو المينا مع واجهة الكثيفة

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or ...

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (&#181;M range) to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) 2. Measurements are based on F&#246;rster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

This manuscript describes how to conduct (single molecule) F&#246;rster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer.

قياس TCR-PMHC التجليد<em&gt; في الموقع</em&gt; باستخدام الميكروسكوب الفحص القائم على الحنق

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The KYF-Pt-NE encapsulates Pt(II) at 10 wt. %, has a diameter of 107 &#177; 27 nm and a negative surface charge. The KYF-Pt-NE is stable in water and in serum, and is biologically active. The conjugation of a fluorophore to KYF allows the synthesis of a fluorescent nanoemulsion that is suitable for biological imaging. The synthesis of the nanoemulsion is performed in an aqueous environment, and the KYF-Pt-NE forms via self-assembly of a short KYF peptide and an oleic acids-platinum(II) conjugate. The self-assembly process depends on the temperature of the solution, the molar ratio of the substrates, and the flow rate of the substrate addition. Crucial steps include maintaining the optimal stirring rate during the synthesis, permitting sufficient time for self-assembly, and pre-concentrating the nanoemulsion gradually in a centrifugal concentrator.

This protocol describes an efficient method to synthesize a nanoemulsion of an oleic acids-platinum(II) conjugate stabilized with a lysine-tyrosine-phenylalanine (KYF) tripeptide. The nanoemulsion forms under mild synthetic conditions via self-assembly of the KYF and the conjugate.

نانومولسيون تريبيبتيدي استقرت حمض الأولييك

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The ...

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 ...

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically been studied in vivo in animal models showing characteristic patterns of cell migration. However, in vivo models have several limitations, including intercellular mediators that are difficult to access and analyze, as well as the inability to directly analyze human neutrophils. Because of these limitations, there is a need for an in vitro platform that studies swarming with human neutrophils and provides easy access to the molecular signals generated during swarming. Here, a multistep microstamping process is used to generate a bioparticle microarray that stimulates swarming by mimicking an in vivo infection. The bioparticle microarray induces neutrophils to swarm in a controlled and stable manner. On the microarray, neutrophils increase in speed and form stable swarms around bioparticle clusters. Additionally, supernatant generated by the neutrophils was analyzed and 16 proteins were discovered to have been differentially expressed over the course of swarming. This in vitro swarming platform facilitates direct analysis of neutrophil migration and protein release in a reproducible, spatially controlled manner.

This protocol generates bioparticle microarrays that provide spatially controlled neutrophil swarming. It provides easy access to the mediators that neutrophils release during migration and allows for quantitative imaging analysis.

الجسيمات الحيوية Microarrays للتحليل الكيميائي والجزيئي للالعدلة البشرية يحتشدون في المختبر

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...

Lipidico Injection Protocol for Serial Crystallography Measurements at the Australian Synchrotron

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built high viscous injector, Lipidico, as part of the macromolecular crystallography (MX2) beamline to measure large numbers of small crystals at room temperature. The goal of this technique is to enable crystals to be grown/transferred to&#160;glass syringes to be used directly in the injector for serial crystallography data collection. The advantages of this injector include the ability to respond rapidly&#160;to changes in the flow rate without interruption of the stream. Several limitations for this high viscosity injector (HVI) exist which include a restriction on the allowed sample viscosities to &#62;10 Pa.s. Stream stability can also potentially be an issue depending on the specific properties of the sample. A detailed protocol for how to set up samples and operate the injector for serial crystallography measurements at the Australian synchrotron is presented here. The method demonstrates preparation of the sample, including the transfer of lysozyme crystals into a high viscous media (silicone grease), and the operation of the injector for data collection at MX2.

The goal of this protocol is to demonstrate how to prepare serial crystallography samples for data collection on a high viscous injector, Lipidico, recently commissioned at the Australian synchrotron.

بروتوكول حقن الدهون لقياسات البلورات التسلسلية في السنكروترون الأسترالي

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built ...

Molecular Spring Constant Analysis by Biomembrane Force Probe Spectroscopy

A biomembrane force probe (BFP) has recently emerged as a native-cell-surface or in situ dynamic force spectroscopy (DFS) nanotool that can measure single-molecular binding kinetics, assess mechanical properties of ligand-receptor interactions, visualize protein dynamic conformational changes and more excitingly elucidate receptor mediated cell mechanosensing mechanisms. More recently, BFP has been used to measure the spring constant of molecular bonds. This protocol describes the step-by-step procedure to perform molecular spring constant DFS analysis. Specifically, two BFP operation modes are discussed, namely the Bead-Cell and Bead-Bead modes. This protocol focuses on deriving spring constants of the molecular bond and cell from DFS raw data.

A biomembrane force probe (BFP) is an in situ dynamic force spectroscopy (DFS) technique. BFP can be used to measure the spring constant of molecular interactions on living cells. This protocol presents spring constant analysis for molecular bonds detected by BFP.

الجزيئية الربيع التحليل المستمر من قبل التحليل الطيفي قوة قوة Biomembrane

A biomembrane force probe (BFP) has recently emerged as a native-cell-surface or in situ dynamic force spectroscopy (DFS) nanotool that can measure ...