خلية ثقافة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تزرع الثقافات المخزون الكلى الجنينية البشرية 293 (HEK293) خلايا في 10 سم ، والأطباق الثقافة في &quot;وسيط&quot; ، ويتألف من متوسطة Dulbecco في التعديل النسر (DMEM) تستكمل مع مزيج من المضادات الحيوية (100 وحدة لكل مل من البنسلين وميكروغرام 100 لكل مل من كبريتات ستربتومايسين) و 10 ٪ مصل بقري جنيني (FBS). </li><li style=";text-align:right;direction:rtl"> لإنشاء خلايا للتجربة ، وامتص المتوسط ​​قبالة ، ويتم غسل الخلايا مرتين مع 5 مل من الفوسفات مخزنة المالحة (PBS) حل. </li><li style=";text-align:right;direction:rtl"> ثم تتم إزالة برنامج تلفزيوني واستبدالها 0.6 مل من محلول يحتوي على 0.25 ٪ التربسين. </li><li style=";text-align:right;direction:rtl"> وحضنت الطبق في 37 ˚ مئوية لمدة حوالي 2 دقيقة حتى الخلايا بدأت فصلها. </li><li style=";text-align:right;direction:rtl"> 5.5 مل من المتوسط ​​هو إضافة إلى تحييد التربسين ويتم عد الخلايا مع عدادة الكريات. </li><li style=";text-align:right;direction:rtl"> وتضعف الخلايا إلى 320000 خلية لكل مل والاستغناء عند 0.1 مل لكل بئر في المغلفة متعدد الليزين 96 - جيدا لوحة الثقافة. </li><li style=";text-align:right;direction:rtl"> مكان الطبق في منديل حاضنة الثقافة في 37 ˚ C مع جو CO2 تحتوي على 8.8 ٪ وتنمو لمدة 24 ساعة. </li></ol> Transfections <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> في التحضير للtransfections ، ينبغي النظر فيه كم هي متماثلة التي يتعين القيام بها في الظروف التجريبية وما مبالغ البلازميد سوف تكون transfected. نقوم بشكل روتيني في جميع الظروف وثلاث نسخ transfect مجموعه من 50 إلى 100 نانوغرام من الحمض النووي البلازميد لكل بئر. وينبغي أن يمزج البلازميد على الأقل مراسل بناء معربا عن يراعة luciferase والتحكم البلازميد luciferase Renilla من التعبير عن مروج التأسيسية. </li><li style=";text-align:right;direction:rtl"> وpipetted حلول البلازميد في أنابيب microcentrifuge 1.5 مل وتستكمل مع 10 ميكرولتر لكل عينة من المصل DMEM حرة وخالية من المضادات الحيوية. على سبيل المثال ، إذا كان 20 بئرا لتكون هي كل transfected 50 ميكروغرام مع الحمض النووي ، إضافة إلى 200 DMEM ميكروليتر DNA ميكروغرام 1. </li><li style=";text-align:right;direction:rtl"> إلى الحل DMEM / إضافة 3 ميكروليتر DNA &quot;العابر 293 كاشف&quot; (Mirus) في الحمض النووي ميكروغرام. مزيج من قبل pipetting ارتفاعا وانخفاضا ، والسماح للعينات تقف في درجة حرارة الغرفة لمدة لا تقل عن 10 دقيقة. </li><li style=";text-align:right;direction:rtl"> خلال هذه الفترة ، وإزالة وسائل الإعلام من 96 لوحة والثقافة ، وكذلك استبدال 90 ألف ميكرولتر من متوسطة جديدة </li><li style=";text-align:right;direction:rtl"> إضافة إلى كل جانب 10 ميكرولتر من الخليط DMEM 293 / DNA / العبور. </li><li style=";text-align:right;direction:rtl"> مكان الطبق في منديل حاضنة الثقافة في 37 ˚ C مع جو CO2 تحتوي على 8.8 ٪ وتنمو لمدة 24 ساعة. </li></ol> البلعمة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد المعلقات المحتوية B المتوسطة (خليط من 01:01 DMEM ولحم الخنزير والمتوسطة بالاضافة الى المضادات الحيوية F12 و 10 ٪ FBS) ، بالإضافة إلى 0.75 ميكرون ، الخرز اللاتكس عند مستوى الصفر ، إلى 1 مغ لكل مليلتر. </li><li style=";text-align:right;direction:rtl"> إزالة وسائل الإعلام من 96 لوحة والثقافة ، وكذلك استبدال بنسبة 0.1 مل من الايقاف في حبة متوسطة B. </li><li style=";text-align:right;direction:rtl"> تدور في لوحة لمدة 2 دقيقة في 1000 × ز </li><li style=";text-align:right;direction:rtl"> ثم يتم استزراع خلايا في 37 ˚ مئوية لمدة 6-16 ساعات. تبعا للظروف والمروج المستخدمة في محرك الأقراص يمكن الكشف luciferase ، مراسل الجين زادت التعبير بعد 1-3 ساعات. </li><li style=";text-align:right;direction:rtl"> في نفس الحالات قد يكون من الضروري لاحتضان الخلايا مع حبات لمدة 30 إلى 60 دقيقة ، ثم نفذ 2 يغسل مع برنامج تلفزيوني لإزالة الخرزات الفردية ، وإضافة جديدة وسائل الاعلام قبل تفرخ الخلايا لفترات إضافية من الزمن. </li></ol> الفحص المزدوج GloTM Luciferase <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ملاحظة : لدينا بروتوكول لاستخدام هذه المجموعة يختلف في بعض النواحي من تلك الموصى بها من قبل الشركة المصنعة. وقد تم تعديل ذلك لتقليل كمية من الكواشف المطلوبة لكل عينة. </li><li style=";text-align:right;direction:rtl"> يعد حل DTT M 1 ، تقسيمها إلى aliquots صغيرة متعددة وتخزينها في -20 جيم ˚ </li><li style=";text-align:right;direction:rtl"> تعد منطقة عازلة تحلل تحتوي على 20 ملي تريس ، حمض الهيدروكلوريك (7.8 درجة الحموضة) ، و 10 ٪ (V / V) الجلسرين ، و 0.5 ٪ (V / V) تريتون X - 100. قبل الاستخدام ، قسامة مبلغ اللازمة للتجربة وإضافة 1 مل من ميكرولتر في DTT M 1 زائد البروتياز كوكتيل مثبط للتركيز النهائي من 0.5 إلى 1X. لا إعادة استخدام الحل DTT متبقية. </li><li style=";text-align:right;direction:rtl"> عند استخدام طقم ثنائي GloTM للمرة الأولى ، إعداد الكاشف (اليراع) Luciferase بنقل محتويات زجاجة واحدة من الاحتياطي Luciferase المزدوجة GloTM (بشرط مع عدة) لزجاجة واحدة من الركيزة Luciferase المزدوجة GloTM (شريطة مع عدة). تقسيم الكاشف Luciferase غير المستخدمة إلى 10 ميلي aliquots وتخزينها في -20 جيم ˚ </li><li style=";text-align:right;direction:rtl"> إزالة لوحة 96 - جيدا من نسيج الثقافة الحاضنة ووضعه على الجليد. إزالة وسائل الاعلام والطموح ، إضافة 40 ميكرولتر لكل بئر من تحلل العازلة ، ثم يترك لوحة على الجليد لمدة 30 دقيقة. </li><li style=";text-align:right;direction:rtl"> في كامد ، أبيض 96 - جيدا لوحة (OptiplateTM - 96 ، رقم الكتالوج بيركن إلمر 6005290) قسامة 15 ميكرولتر لكل بئر من Luciferase الكاشف ، ثم إضافة 15 ميكرولتر من lysate الخلية. </li><li style=";text-align:right;direction:rtl"> احتضان لوحة في درجة حرارة الغرفة لمدة 10 دقيقة ثم قراءة التلألؤ في microplate القارئ. </li><li style=";text-align:right;direction:rtl"> على الفور قبل استخدامه ، وإعداد مبلغ مناسب من Renilla حل الركيزة luciferase بإضافة جزء 1 المزدوجة وإيقاف GloTM بالشبكة ®؛ الكاشف (بشرط مع عدة) إلى 100 جزء المزدوجة وإيقاف GloTM بالشبكة ® العازلة (بشرط مع عدة). </li><li style=";text-align:right;direction:rtl"> إضافة إلى كل جانب 15 ميكرولتر لكل بئر من الحل الركيزة luciferase Renilla ، يحضن في درجة حرارة الغرفة لمدة 10 دقيقة ثم قياس التألق مرة أخرى. </li></ol>

The authors have nothing to disclose.

studying membrane biogenesis with a luciferase-based reporter gene assay

لدراسة مسارات مختلفة من التنسيق توليف الدهون أثناء نشوء حيوي غشاء من المفيد للعمل مع نظام تجريبي حيث نشوء حيوي الغشاء يحدث بسرعة وبطريقة محرض. لقد وجدنا أن البلعمة من الخرز اللاتكس هو عملي لهذه الأغراض كخلايا توليف الدهون بسرعة لتجديد الغشاء غشاء برك كما فقدت التفاف المواد خلال الإحاطة الجسيمات. هنا ، نحن تصف إجراءات لدراسة التغيرات في البلعمة التي يسببها التعبير الجيني مع نهج مراسل الجين luciferase المستندة إلى استخدام مزدوج بالشبكة نظام الفحص Luciferase من Promega.

Watch this Scientific Journal Video about Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay at JoVE.com

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

هنا ، نحن تصف إجراءات لدراسة التغيرات في البلعمة التي يسببها التعبير الجيني مع نهج مراسل الجين luciferase المستندة إلى استخدام مزدوج GloTM نظام الفحص Luciferase من Promega.

دراسة النشوء الأحيائي الأغشية مع الفحص الجيني مراسل Luciferase المستندة

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

studying-membrane-biogenesis-with-luciferase-based-reporter-gene

Research

JoVE Journal

Biology

12.8K Views.  Harvard. هنا ، نحن تصف إجراءات لدراسة التغيرات في البلعمة التي يسببها التعبير الجيني مع نهج مراسل الجين luciferase المستندة إلى استخدام مزدوج GloTM نظام الفحص Luciferase من Promega.

Video: Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 &mu;m wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37&deg;C, 5% CO2). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

جهاز ميكروفلويديك مع أنماط Groove لدراسة السلوك الخليوي

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

باستخدام الجينات Electroporation MXcell بولسير النظام لTransfect الخلايا الأولية ذات الكفاءة العالية

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

الأزهار وتراجع تحويل thaliana Arabidopsis لتفحص pTSO2 : β غلوكورونيداز التعبير مراسل جين

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

الأجرعية بوساطة الفيروسات إسكات الجينات المستحثة الفحص في القطن

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...

Imaging Plasma Membrane Deformations With pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress.
Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or&#160;diD. DiD intercalates in the membrane plane with a &#34;fixed&#34; orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561&#160;nm laser (p-pol, s-pol). A 488&#160;nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

التصوير البلازما غشاء التشويهات مع pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard approach to determining intracellular protein degradation relies on biochemical assays for following the kinetics of protein decline. Such methods are often laborious and time consuming and therefore not amenable to experiments aimed at assessing multiple substrates and degradation conditions. As an alternative, cell growth-based assays have been developed, that are, in their conventional format, end-point assays that cannot quantitatively determine relative changes in protein levels.
Here we describe a method that faithfully determines changes in protein degradation rates by coupling them to yeast cell-growth kinetics. The method is based on an established selection system where uracil auxotrophy of URA3-deleted yeast cells is rescued by an exogenously expressed reporter protein, comprised of a fusion between the essential URA3 gene and a degradation determinant (degron). The reporter protein is designed so that its synthesis rate is constant whilst its degradation rate is determined by the degron. As cell growth in uracil-deficient medium is proportional to the relative levels of Ura3, growth kinetics are entirely dependent on the reporter protein degradation.
This method accurately measures changes in intracellular protein degradation kinetics. It was applied to: (a) Assessing the relative contribution of known ubiquitin-conjugating factors to proteolysis (b) E2 conjugating enzyme structure-function analyses (c) Identification and characterization of novel degrons. Application of the degron-URA3-based system transcends the protein degradation field, as it can also be adapted to monitoring changes of protein levels associated with functions of other cellular pathways.

Here we describe a robust biological assay for quantifying the relative rate of proteolysis by the ubiquitin-proteasome system. The assay readout is yeast growth rate in liquid culture, which is dependent on the cellular levels of a reporter protein comprising a degradation signal fused to an essential metabolic marker.

الفحص النمو القائم على مراسل لتحليل منهجي من تدهور البروتين

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard ...

Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been described, certain types of cells are resistant to many of these methods and yield low transfection efficiency1, potentially hindering research in those cell types. In this protocol, we present an optimized transfection procedure to introduce luciferase reporter genes as a plasmid DNA into the RAW264.7 macrophage cell line. Two different types of transfection reagents (lipid-based and polyamine-based) are described, and important notes are given throughout the protocol to ensure that the RAW264.7 cells are minimally altered by the transfection procedure and any experimental data obtained are the direct results of the experimental treatment. While transfection efficiency may not be higher compared to other transfection methods, the described procedure is robust enough for detecting luciferase signal in RAW264.7 without changing the physiological response of the cells to stimuli.

Transfection into the macrophage cell line, RAW264.7, is difficult due to the cell&#8217;s natural response against foreign materials. We described here a gentle yet robust procedure for transfecting luciferase reporter genes into RAW264.7 cells.

بنقل العدوى إلى خلايا RAW264.7 مع luciferase المراسل مراسل جين

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

تصور التردد Stromule مع مضان المجهر

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and develops most often in the setting of immunosuppression. Despite decades of research, optimal treatment of KS remains poorly defined and clinical outcomes are especially unfavorable in resource-limited settings. KS lesions are driven by pathological angiogenesis, chronic inflammation, and oncogenesis, and various in vitro cell culture models have been developed to study these processes. KS arises from KSHV-infected cells of endothelial origin, so EC-lineage cells provide the most appropriate in vitro surrogates of the spindle cell precursor. However, because EC have a limited in vitro lifespan, and as the oncogenic mechanisms employed by KSHV are less efficient than those of other tumorigenic viruses, it has been difficult to assess the processes of transformation in primary or telomerase-immortalized EC. Therefore, a novel EC-based culture model was developed that readily supports transformation following infection with KSHV. Ectopic expression of the E6 and E7 genes of human papillomavirus type 16 allows for extended culture of age- and passage-matched mock- and KSHV-infected EC and supports the development of a truly transformed (i.e., tumorigenic) phenotype in infected cell cultures. This tractable and highly reproducible model of KS has facilitated the discovery of several essential signaling pathways with high potential for translation into clinical settings.

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is a tumor induced by infection with the oncogenic virus human herpesvirus-8/KS herpesvirus (HHV-8/KSHV). The endothelial cell culture model described here is uniquely suited for studying the mechanisms by which KSHV transforms host cells.

نموذج في المختبر لدراسة التحول الخلوي من هربس كابوزي ساركوما

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and ...