وأيد هذا العمل من قبل عدد يلكوم ترست منحة [077108] منحت لGJW.

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عندما لاحظ الخلايا الحية في الجسم الحي، والأغشية البلازما من معرض السلوكيات ديناميكية للغاية: مد وعمليات غشاء التراجع لأنها الاتصال والتواصل مع 2 جيرانهم. وقد تطورت التفاعلات الجسدية بين بروتينات مستقبلات غشاء جزءا لا يتجزأ من الذي توسط في العديد من هذه الاتصالات ان يكون ضعيفا جدا بحيث تسمح لهذا السلوك متحركة جدا. وتشير التقديرات إلى أن قوة التفاعل أحادى على أنه ضعيف إلى 50 ميكرومتر يمكن أن يكون كافيا لدفع التفاعل العفوي بكثافة مستقبلات الفسيولوجية (9) الذي يجعل الكشف عن التفاعلات رواية صعبة للغاية 2،10. طريقة AVEXIS الموصوفة هنا يوفر طريقة حساسة للكشف عن البروتين خارج الخلية عابر: تفاعلات البروتين التي يمكن تنفيذها بطريقة متدرجة مع انخفاض معدل إيجابية كاذبة. في حين وضعت أساليب للتحجيم أخرى للكشف عن التفاعلات خارج الخلية 11-15،ميزة واحدة من AVEXIS هي التي تم كميا المعلمات التجريبية مثل الطعم والأنشطة فريسة للكشف عن حتى أضعف من التفاعلات (ر 1/2 ≤ 0.1 ق) مع الحفاظ على انخفاض معدل إيجابية كاذبة 3. وبالإضافة إلى ذلك، مكنت إعداد نموذج مبسط ومتين إجراءات هذا الفحص إلى أن تنفذ على نطاق أوسع بكثير من فحوصات أخرى والتي وصفناها في الآونة الأخيرة على شاشة تفاعل منهجي تقدر بأكثر من التفاعلات المحتملة 16500 ~ 16. لا يمكن أن يؤديها للفحص في أي من البروتين الذي كان من الممكن للتعبير عن ذوبان قطعة بروتين المؤتلف. وهذا يشمل ذلك البروتينات التي تحتوي على الببتيد إشارة N-محطة مثل النوع الأول، GPI مرتبطة وتفرز بروتينات. لقد قمنا بتصميم مؤخرا مجموعة من النواقل التي تمكن الآن لنا للتعبير عن نوع من البروتينات الثاني والطعم 17. الفحص ليست عادة مناسبة للبروتينات غشاء المتعددة الدخول مثل نقل أيونق أو مضخات لأنها من غير المرجح أن تكون مطوية بشكل صحيح خارج سياق غشاء البلازما. وقد طبقنا هذا على مجموعة متنوعة من نظم مختلفة وأعربوا عن بنجاح البروتينات خارج الخلية من الزرد 3،16،18، والبشرية، والفأرة P. المنجلية لشاشات التفاعل. من حيث الموارد اللازمة لانشاء شاشة AVEXIS، وجدنا ان عنق الزجاجة أهمية هو اختيار، والبناء، والتسلسل الكامل للالبلازميدات التعبير. ويمكن في هذا الجانب من أسلوب يستغرق فترة تصل الى عام لجعل ~ 100 الطعم والبنى فريسة، ولكن، مع انخفاض تكاليف التركيب الجيني، نؤيد الآن تجاريا الاستعانة بمصادر خارجية في هذا الجانب من المشروع. نظام التعبير الثدييات جنبا إلى جنب مع كبير يهز الأنسجة حاضنة الثقافة (ونحن استخدام خلية INFORS Multitron HT) تمكن الباحث وحيد للتعبير عن وتطبيع ما يصل الى 100 ~ الطعم والبروتينات فريسة في الشهر. مرة واحدة ويتم إنتاج البروتينات وتطبيع، قcreening للتفاعل سريع مع ما يصل إلى 12 لوحات 96-جيدا يتم معالجتها بشكل ملائم في اليوم الواحد. المعدات مفصل إلا أننا قد وضعت لتسهيل انتاج هذا العدد الكبير من البروتينات هو هواء مضغوط يحركها المكبس الذي يستخدم لتمرير ما يصل الى خمسة supernatants من خلال مرشح 0.22μm بشكل متواز. الطبقة الرئيسية من التفاعلات التي قد غاب بالمقارنة مع التفاعلات المتوقعة من الأدب (كاذبة سلبيات) هي التفاعلات مقتصر على المطابق، على الرغم من أن يمكن الكشف عن بعض من هذه التفاعلات 3. في الحالات التي لم يتم الكشف عن التفاعلات المتوقعة، نعتقد أن هذا يرجع إلى تشكيل التفاعلات مقتصر على المطابق من البروتينات فريسة (أ فريسة &quot;اخفاء&quot; التأثير) الذي يحول دون مزيد من التفاعل ثم مع البروتينات الطعم يجمد. التردد الذي يتم الكشف عن التفاعلات يعتمد على محتوى مكتبة بروتين يجري فرزهم. كدليل للقارئ، وشاشة من&gt; 30، نتج 000 التفاعلات داخل الفصيلة المناعي الزرد في 188 ايجابيات - تردد 0.6٪ 3،16،18. شيك سريع جدا التي يمكن القيام بها للتحقق من أي إصابات إيجابية تتمثل في تحديد ما إذا كان يمكن الكشف عن تفاعل التوجهات في الطعم والفريسة على حد سواء وهذا هو، ويمكن الكشف عن التفاعل نفسه بغض النظر عن ما إذا كانت ملزمة البروتينات موجودة إما الطعم أو فريسة. كلما لاحظنا هذا السلوك تبادل، وبعد ذلك تم الربط أظهرت أن تكون محددة من خلال إظهار ملزم تشبع مباشرة لتنقية البروتينات باستخدام سطح صدى مأكل. وينبغي التأكيد على أن زيادة الطمع، لأننا ملزم من قبل pentamerising مصطنع البروتينات فريسة، ونحن لا نعتبر فحص مناسبة للمقارنة كميا قوة التفاعل. للحصول على المعلمات ملزم البيوفيزيائية من التفاعلات نستخدم البروتينات أحادى وسطح صدى مأكل. أخيرا، مرة واحدة وقد تم التعرف على التفاعل، ومرحباغ طبيعة الإنتاجية للفحص يجعل من السهل نسبيا على التكيف مع الفحص للكشف عن المواد الكاشفة مثل الأجسام المضادة أو الجزيئات الصغيرة التي تعمل على منع التفاعل.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> القط. عدد </td><td> تعليقات </td></tr><tr><td> حرة وسائل الاعلام </td><td> إينفيتروجن </td><td> 12338018 </td><td></td></tr><tr><td> Nitrocefin </td><td> Calbiochem </td><td> 484400 </td><td></td></tr><tr><td> لوحات microtitre SA-المغلفة </td><td> نونك </td><td> 734-1284 </td><td></td></tr><tr><td> OX68 الأضداد </td><td> AbDSerotec </td><td> MCA1022R </td><td></td></tr><tr><td> غسيل الأنابيب </td><td> ثقب </td><td> PN68100 </td><td></td></tr><tr><td> بوليميكسين باء </td><td> سيغما </td><td> P4932 </td><td></td></tr><tr><td> مد البيوتين </td><td> سيغما </td><td> B4639-5G </td><td></td></tr><tr><td> الركيزة-104 </td><td> سيغما </td><td> 1040-506 </td><td></td></tr><tr><td> Vivaspin-20 مكثفات الطرد المركزي </td><td> سارتوريوس </td><td> VS2002 </td><td></td></tr><tr><td> 0.22 ميكرون مرشحات </td><td> Nalgene </td><td> 190-2520 </td><td></td></tr></tbody></table>

avidity-based extracellular interaction screening (avexis) for the scalable detection of low-affinity extracellular receptor-ligand interactions

بروتين خارج الخلية: التفاعلات بين البروتينات أو تفرز بروتينات غشاء المربوطة حاسمة بالنسبة لكل من التماسك بدء الاتصالات بين الخلايا، وضمان في الكائنات متعددة الخلايا. وتوقع لتشكيل البروتينات يتم ترميز التفاعلات خارج الخلية بمقدار الربع تقريبا من الجينات البشرية 1، ولكن على الرغم من أهميتها وفرة، وغالبية هذه البروتينات وقد وثقت لا شريك ملزم. في المقام الأول، وهذا يرجع إلى استعصاء على البيوكيميائية: بروتينات غشاء جزءا لا يتجزأ من الصعب solubilise في التشكل وطنهم وتحتوي على تعديلات posttranslational الهيكلية الهامة. أيضا، وتتميز في كثير من الأحيان الانتماءات تفاعل بين البروتينات المستقبلة من قبل القوة تفاعل منخفضة للغاية (نصف حياة &lt;1 2) النافية الكشف عنها مع العديد من الشائع استخدام أساليب إنتاجية عالية 2. هنا، نحن تصف فحص، AVEXIS (الطمع القائم على EXtracelluلار التفاعل الشاشة) أن يتغلب على هذه التحديات التقنية التي تمكن من الكشف عن تفاعلات البروتين ضعيف جدا (ر 1/2 ≤ 0.1 ثانية) مع انخفاض معدل إيجابية كاذبة 3. وينفذ عادة الفحص في شكل إنتاجية عالية لتمكين فحص المنتظم لعدة آلاف من التفاعلات في شكل لوحة microtitre مناسب (الشكل 1). لأنه يعتمد على إنتاج بروتين قابل للذوبان المكتبات المؤتلف التي تحتوي على شظايا ectodomain من مستقبلات سطح الخلية أو البروتينات يفرز من خلاله للكشف عن التفاعلات، وبالتالي، فإن هذا النهج هو مناسبة لنوع أنا، من النوع الثاني، GPI المرتبطة مستقبلات سطح الخلية وتفرز البروتينات ولكن ليس للبروتينات غشاء المتعددة الدخول مثل القنوات الأيونية أو النقل. ويتم إنتاج بروتين المؤتلف المكتبات باستخدام مريحة وعلى مستوى رفيع نظام التعبير الثدييات 4، لضمان أن تعديلات هامة posttranslational مثل glycosylaتضاف نشوئها وثاني كبريتيد السندات. تفرز البروتينات المؤتلف وأعرب في المتوسط ​​والتي تنتج في شكلين: الطعم المعقدة البيروكسيديز التي يمكن الحصول عليها في مرحلة streptavidin المغلفة صلبة مناسبة للفحص، وpentamerised انزيم الموسومة فريسة (β-اكتاماز). يتم تقديم الطعم والبروتينات فريسة لبعضها البعض على نحو ثنائي للكشف عن التفاعلات المباشرة بينهما، على غرار ELISA التقليدية (الشكل 1). يتحقق pentamerisation من البروتينات في الفريسة من خلال سلسلة ببتيد من المصفوفة بروتين غضروف oligomeric (شركات) ويزيد من تركيز المحلية لectodomains مما يوفر مكاسب كبيرة لتمكين الطمع إلى أن يتم الكشف عن التفاعلات حتى عابر جدا. من قبل تطبيع أنشطة الطعم على حد سواء وفريسة لمستويات محددة سلفا قبل الفرز، وأظهرنا أنه يمكن الكشف عن وجود تفاعلات أحادى نصف حياة 0.1 ثانية مع انخفاض معدلات إيجابية كاذبة 3.

Watch this Scientific Journal Video about Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions at JoVE.com

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS هو بروتين إنتاجية عالية مقايسة التفاعل المتقدمة إلى الشاشة بشكل منتظم لرواية أزواج مستقبلات يجند خارج الخلية المشاركة في عمليات التعرف على الخليوي. وهي مصممة خصيصا للكشف عن بروتين تفاعلات عابرة يصعب تحديد استخدام غيرها من النهج إنتاجية عالية.

الطمع القائم على فحص التفاعل خارج الخلية (AVEXIS) للكشف قابلة منخفضة تقارب التفاعلات مستقبلات يجند خارج الخلية

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

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Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

21.4K Views.  Wellcome Trust Sanger Institute. AVEXIS هو بروتين إنتاجية عالية مقايسة التفاعل المتقدمة إلى الشاشة بشكل منتظم لرواية أزواج مستقبلات يجند خارج الخلية المشاركة في عمليات التعرف على الخليوي. وهي مصممة خصيصا للكشف عن بروتين تفاعلات عابرة يصعب تحديد استخدام غيرها من النهج إنتاجية عالية.

Video: Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.
An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.
Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)1 (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself2, and is amenable to high-throughput analyses3. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.
OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes4. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase5, 11, 12, 13, xylan using an endo-&beta;-(1-4)-xylanase 6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase 8. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined 9.

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

أليغو التنميط قداس (OLIMP) من السكريات خارج الخلية

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

تقنيات لتحليل خارج الخلية الحويصلات عن طريق التدفق الخلوي

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers such as epithelia also form a barrier that separates the inside from the outside or different compartments of multicellular organisms. A key feature of these barriers is the differential distribution of ions across cell membranes or cell layers. Two properties allow this distribution: 1) membranes and epithelia display selective permeability to specific ions; 2) ions are transported through pumps across cell membranes and cell layers. These properties play crucial roles in maintaining tissue physiology and act as signaling cues after damage, during repair, or under pathological condition. The ion-selective self-referencing microelectrode allows measurements of specific fluxes of ions such as calcium, potassium or sodium at single cell and tissue levels. The microelectrode contains an ionophore cocktail which is selectively permeable to a specific ion. The internal filling solution contains a set concentration of the ion of interest. The electric potential of the microelectrode is determined by the outside concentration of the ion. As the ion concentration varies, the potential of the microelectrode changes as a function of the log of the ion activity. When moved back and forth near a source or sink of the ion (i.e. in a concentration gradient due to ion flux) the microelectrode potential fluctuates at an amplitude proportional to the ion flux/gradient. The amplifier amplifies the microelectrode signal and the output is recorded on computer. The ion flux can then be calculated by Fick&#8217;s law of diffusion using the electrode potential fluctuation, the excursion of microelectrode, and other parameters such as the specific ion mobility. In this paper, we describe in detail the methodology to measure extracellular ion fluxes using the ion-selective self-referencing microelectrode and present some representative results.

Transporters in cell membranes allow differential segregation of ions across cell membranes or cell layers and play crucial roles during tissue physiology, repair and pathology. We describe the ion-selective self-referencing microelectrode that allows the measurement of specific ion fluxes at single cells and tissues in vivo.

قياس خارج الخلية ايون تدفقاتها باستخدام ايون انتقائية مسرى مكروي تقنية الرجوع الذاتي

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers ...

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

كفاءة الثدييات التعبير خلية وخطوة واحدة خارج الخلية تنقية البروتينات السكرية لبلورة

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...

Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix biology, there are still major gaps related to how the matrix is formed, how physiological and pathological remodeling differ, and most importantly how matrix dynamics might be manipulated to promote healing and inhibit fibrosis. There is currently no treatment option for controlling, preventing, or reversing cardiac fibrosis. Part of the reason is likely the sheer complexity of cardiac scar formation, such as occurs after myocardial infarction to immediately replace dead or dying cardiomyocytes. The extracellular matrix itself participates in remodeling by activating resident cells and also by helping to guide infiltrating cells to the defunct lesion. The matrix is also a storage locker of sorts for matricellular proteins that are crucial to normal matrix turnover, as well as fibrotic signaling. The matrix has additionally been demonstrated to play an electromechanical role in cardiac tissue. Most techniques for assessing fibrosis are not qualitative in nature, but rather provide quantitative results that are useful for comparing two groups but that do not provide information related to the underlying matrix structure. Highlighted here is a technique for visualizing cardiac matrix ultrastructure. Scanning electron microscopy of decellularized heart tissue reveals striking differences in structure that might otherwise be missed using traditional quantitative research methods.

Shown here is a method for visualizing extracellular matrix ultrastructure in decellularized cardiac tissues.

المجهر الإلكتروني من الأنسجة متآكلة إلى تصور مصفوفة خارج الخلية

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix ...

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.
Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.

والسريعة، طريقة قابلة للعزل، دراسة وظيفية، وتحليل المصفوفة خارج الخلية المشتقة من خلية

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

إنشاء درجة الحموضة خارج الخلية الحمضية منظومة الثقافة

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...

A BW Reporter System for Studying Receptor-Ligand Interactions

Interactions between receptors and ligands constitute a fundamental biological process. However, direct experiments with cells that express the native receptor and the ligand are challenging since the ligand of a specific receptor may be unknown and experimental procedures with the native ligand can be technically complicated. To address these obstacles, we describe a reporter system to detect the binding and activation of a specific receptor by a ligand of interest. In this reporter system, the extracellular domain of a specific receptor is conjugated to mouse CD3&#950; and this chimeric protein is then expressed in mouse BW cells. These transfected BW cells can then be incubated with different targets (e.g., cells or antibodies). Activation of a transfected receptor leads to the secretion of mouse interleukin-2 (mIL-2) which can be detected by enzyme-linked immunosorbent assay (ELISA). This reporter system has the advantages of being sensitive and specific to a single receptor. In addition, the activation level of a specific receptor can easily be quantified and can be used even in cases where the ligand of the receptor is unknown. This system has been implemented successfully in many of our studies to characterize receptor-ligand interactions. We have recently employed this system to study the activation of human Fc&#947; receptors (Fc&#947;Rs) by different monoclonal anti-CD20 antibodies in clinical use.

This protocol describes how to establish a reporter system that can be used to identify and quantify receptor-ligand interactions.

نظام مراسل الحرب البيولوجية لدراسة التفاعلات مستقبلات-يجند

Interactions between receptors and ligands constitute a fundamental biological process. However, direct experiments with cells that express the native ...

Nanoparticle Tracking Analysis for the Quantification and Size Determination of Extracellular Vesicles

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving area of research. There are many different methods for EV isolation, each with distinct advantages and disadvantages that affect the downstream yield and purity of EVs. Thus, characterizing the EV prep isolated from a given source by a chosen method is important for interpretation of downstream results and comparison of results across laboratories. Various methods exist for determining the size and quantity of EVs, which can be altered by disease states or in response to external conditions. Nanoparticle tracking analysis (NTA) is one of the prominent technologies used for high-throughput analysis of individual EVs. Here, we present a detailed protocol for quantification and size determination of EVs isolated from mouse perigonadal adipose tissue and human plasma using a breakthrough technology for NTA representing major advances in the field. The results demonstrate that this method can deliver reproducible and valid total particle concentration and size distribution data for EVs isolated from different sources using different methods, as confirmed by transmission electron microscopy. The adaptation of this instrument for NTA will address the need for standardization in NTA methods to increase rigor and reproducibility in EV research.

We demonstrate how to use a novel nanoparticle tracking analysis instrument to estimate the size distribution and total particle concentration of extracellular vesicles isolated from mouse perigonadal adipose tissue and human plasma.

تحليل تتبع الجسيمات النانوية لتحديد كمي وحجم الحويصلات خارج الخلية

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving ...

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

There is a need for practical assays to visualize and quantify the cells' extracellular vesicle (EV) uptake. EV uptake plays a role in intercellular communication in various research fields; cancer biology, neuroscience, and drug delivery. Many EV uptake assays have been reported in the literature; however, there is a lack of practical, detailed experimental methodology. EV uptake can be assessed by fluorescently labeling EVs to detect their location within cells. Distinguishing between internalized EVs in cells and the superficial EVs on cells is difficult, yet critical, to accurately determine the EV uptake. Therefore, an assay that efficiently quantifies EV uptake through three-dimensional (3D) fluorescence confocal microscopy is proposed in this work. Fluorescently labeled EVs were prepared using a nano-filtration-based microfluidic device, visualized by 3D confocal microscopy, and then analyzed through advanced image-processing software. The protocol provides a robust methodology for analyzing EVs on a cellular level and a practical approach for efficient analysis.

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

Extracellular vesicles (EVs) contribute to cellular biology and intercellular communications. There is a need for practical assays to visualize and quantify EVs uptake by the cells. The current protocol proposes the EV uptake assay by utilizing three-dimensional fluorescence imaging via confocal microscopy, following EV isolation by a nano-filtration-based microfluidic device.

خارج الخلية Vesicle امتصاص المقايسة عبر تحليل التصوير المجهر Confocal

There is a need for practical assays to visualize and quantify the cells' extracellular vesicle (EV) uptake. EV uptake plays a role in intercellular ...