وأيد هذا العمل جزئيا من صندوق هورويتز إيشايا.

IL - 8 مقايسة : <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وشملت خليط التفاعل المكونات الأربعة التالية من حيث الحجم النهائي 100 ميكرولتر من مقايسة العازلة في تركيزات كل منهما. 10 ميكروليتر من الخرز المغناطيسي مع الأضداد في التقاط حبات 100 / ميكرولتر تركيزات النهائي ، و 2 ميكرولتر من IL - 8 biotinylated الأضداد في التركيز النهائي nano-gram/μl 1 ، 1 ميكرولتر من بروتين فلوري streptavidin بتركيز 20 nano-gram/μl النهائي ، و 1 ميكرولتر من IL - 8 المستهدف عند 0.48 pico-gram/μl. تضاف المكونات واحدا تلو الآخر إلى المخزن المؤقت مقايسة واهتزت لمدة 30 دقيقة ثم. </li><li style=";text-align:right;direction:rtl"> ويتم إعداد رد فعل السيطرة على نفس الطريق دون أن يكون الهدف IL - 8. </li><li style=";text-align:right;direction:rtl"> يتم وضعها في وقت لاحق من دون أي ردود فعل الانفصال أو خطوة الغسيل في cuvettes وتفتيشها باستخدام نظام MMB. </li></ol> MMB النظام : <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ضع كفيت في موقفه بين القطبين الكهرومغناطيسي. </li><li style=";text-align:right;direction:rtl"> العمل الحالي التشكيل وانتظر 30 ثانية للسماح التجميع والتكثيف من الخرز </li><li style=";text-align:right;direction:rtl"> قياس إشارة القفل في مكبر للصوت باستخدام الذبذبات. </li></ol> ممثل النتائج : قدم الفحص البصري من الخرز المجمعة فرق واضح بين التفاعل مع الهدف والتفاعل من دون هدف. في ردود الفعل على كل شيء مع هذا الهدف ، شكلت الخرز واحد ، التجميعية الكثيفة التي انتقلت من وإلى شعاع الليزر بطريقة توحد. ومع ذلك ، في رد فعل كل ذلك من دون هدف ، كانت حبات أقل مجمعة وحركتهم كان أقل توحيد (انظر الشكل 1). <img src="/files/ftp_upload/1935/1935fig1.jpg" alt="الشكل 1" /> الشكل 1 : التفتيش البصري المناعية الساندويتش &quot;(أ) دون هدف IL - 8 (ب) والهدف IL - 8. وتعرض على مدار الساعة التشكيل قطب (الصفراء) وإشارة الناتج PMT (أرجواني) ، في حين كشف 0.48 بيكو غرام IL - 8 المستهدفة في الشكل 1 (أ). تردد التحوير لكل قطب هو في 2 هرتز. لكن ، وكما كان متوقعا من الناحية النظرية ، عند حبات تمرير شعاع ليزر ، ويكشف PMT ضوء الفلورسنت وهناك قمة في انتاج التيار الكهربائي PMT. لذلك ، تردد هو الإستخلاص في 4 هرتز. عندما يتم تغذية إشارة PMT وعلى مدار الساعة ، تضاعف التعديل (في 4 هرتز) للقفل في مكبر للصوت ، في مرحلة حساسة للكشف عن بالكشف عن التزامن والنتائج مع الجهد العالي (انظر الشكل 2). <img src="/files/ftp_upload/1935/1935fig2.jpg" alt="الشكل 2" /> الشكل 2 : (أ) على مدار الساعة التشكيل (الأصفر) ، وإشارة PMT (أرجواني) عند الكشف 0.48 بيكو غرام IL - 8 المستهدفة. (ب) وتأمين التيار الكهربائي أدى إلى مكبر للصوت على اثنين من يمسح مختلفة. لم القفل في مكبر للصوت لا يكشف عن أي إشارة عندما تم اختبار عينة السيطرة. هذه الحقيقة ، جنبا إلى جنب مع الفرق البصرية في التجميع تشير إلى أن نظام MMB يمكن تحديد واضح لوجود IL - 8 المستهدفة.

<ol>
	<li>Danielli, A., Arie, A., Porat, N., Ehrlich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+fluorescent-labeled+probes+at+sub-picomolar+concentrations+by+magnetic+modulation.">Detection of fluorescent-labeled probes at sub-picomolar concentrations by magnetic modulation.</a> Optics Express. 16, 19253-19259 (2008).</li><li>Danielli, A., Porat, N., Arie, A., Ehrlich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+homogenous+detection+of+the+Ibaraki+virus+NS3+cDNA+at+pico-molar+concentrations+by+magnetic+modulation.">Rapid homogenous detection of the Ibaraki virus NS3 cDNA at pico-molar concentrations by magnetic modulation.</a> Biosensors and Bioelectronics. 25, 858-863 (2009).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bio-Rad "Human angiogenesis assay panel". , .</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Instruction+Manual.">Instruction Manual.</a> Bio-Plex Precision Pro Cytokine Assay. , .</li></ol>

باختصار ، لقد أظهرنا أنه يمكن استخدام هذا النظام MMB للكشف عن وجود IL - 8 هدف في تركيزات منخفضة (0.5 بيكو غرام هو الحد من الكشف عن فحوصات دقيقة بيو برو خلوى الصفيف [3،4]). لا يقتصر على قدرة النظام على IL - 8 ، ويمكن استخدامها للكشف عن البروتينات الأخرى. مزايا نظام MMB هي القدرة على ?...

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rapid homogeneous detection of biological assays using magnetic modulation biosensing system

نظام مغناطيسي biosensing التعديل (MMB) [1،2] بسرعة والكشف عن الأهداف الحيوية بشكل متجانس في تركيزات منخفضة دون أي خطوة الغسيل أو الانفصال. عندما IL - 8 الهدف كان حاضرا ، وهو &#39;sandwich&#39; المستندة إلى مقايسة المرفقة مع الخرز المغناطيسي IL - 8 القبض على الضد streptavidin بروتين فلوري جانب طريق مستهدفة IL - 8 و IL - 8 biotinylated الأضداد. وناور حبات المغناطيسي في حركة متذبذبة من خلال تطبيق المجال المغناطيسي بالتناوب التدرج من خلال قطبين الكهرومغناطيسي. ومكثف البروتينات الفلورية ، والتي تعلق على حبات المغناطيسي في المنطقة والكشف عن تحركاتهم داخل وخارج لشعاع الليزر متعامد تنتج إشارة الدوري الفلورسنت التي يتم الكشف demodulated باستخدام متزامن. كان يستخدم سابقا biosensing تعديل النظام المغناطيسي للكشف عن تسلسل الترميز للبروتين الفيروس غير الهيكلية ايباراكي 3 (NS3) DNA التكميلية ([كدنا]) [2]. ويمكن استخدام التقنيات التي أثبتت في هذا العمل الخارجي للتلاعب والتكثيف من الجزيئات لتطبيقات أخرى ، مثل تسليم المخدرات ، إلى جانب مغناطيسيا<em&gt; في الجسم الحي.</em&gt; أو على النقيض من أجل تعزيز<em&gt; في الجسم الحي.</emتطبيقات التصوير&gt;.

Watch this Scientific Journal Video about Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System at JoVE.com

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

ويستخدم نظام مغناطيسي لتعديل biosensing بسرعة ، بحساسية وبساطة كشف المقايسات البيولوجية ، مثل جزيئات الحمض النووي والبروتينات.

متجانسة الكشف السريع عن طريق فحوصات البيولوجي المغناطيسي نظام Biosensing التحوير

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

rapid-homogeneous-detection-biological-assays-using-magnetic

Research

JoVE Journal

Biology

9.6K Views.  Tel Aviv University. ويستخدم نظام مغناطيسي لتعديل biosensing بسرعة ، بحساسية وبساطة كشف المقايسات البيولوجية ، مثل جزيئات الحمض النووي والبروتينات.

Video: Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous mycotoxins in food commodities. In this protocol we show how to fabricate and interrogate an electrochemical competitive Enzyme linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain3 and screen printed electrodes as sensing platform.
Our method aims to determine the total amount of HT-2 and T-2 toxins, mycotoxins belonging to the trichothecenes family and of great concern for human health4. The use of an antibody clone with a cross reactivity of 100% towards HT-2 and T-2 allows to simultaneously detect both toxins with similar sensitivity5.
The first step of our assay is the coating step where we immobilize HT2-KLH conjugate toxin on the surface of magnetic beads. After a blocking step, necessary to avoid non-specific absorptions, the addition of a monoclonal antibody allows the competition between immobilized HT-2 and free HT-2 or T-2 present in the sample or dissolved in a standard solution.
At the end of the competition step, the amount of monoclonal antibody linked to the immobilized HT-2 will be inversely proportional to the amount of toxin in the sample solution.
A secondary antibody labeled with alkaline phosphatase (AP) is used to reveal the binding between the specific antibody and the immobilized HT-2. The final measurement step is performed by dropping an aliquot of magnetic bead suspension, corresponding to a specific sample/standard solution, on the surface of a screen-printed working electrode; magnetic beads are immobilized and concentrated by means of a magnet placed precisely under the screen-printed electrode. After two minutes of incubation between magnetic beads and a substrate for AP, the enzymatic product is detected by Differential Pulse Voltammetry (DPV) using a portable instrument (PalmSens) also able to initiate automatically eight measurements within an interval of few seconds.

ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection

A protocol to detect trichothecenes (mycotoxins of concern for human health) using a newly developed screening method based on a competitive immunochemical method and a final electrochemical detection is demonstrated.

ELIME (انزيم مرتبط المناعي المغناطيسي الكهروكيميائية) طرق الكشف عن السموم الفطرية

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous ...

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

تحليل شفان نجمية طريق التفاعلات في المختبر فحوصات

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Rapid PCR Thermocycling using Microscale Thermal Convection

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable concentration level. But the design of conventional PCR thermocycling hardware, predominantly based on massive metal heating blocks whose temperature is regulated by thermoelectric heaters, severely limits the achievable reaction speed1. Considerable electrical power is also required to repeatedly heat and cool the reagent mixture, limiting the ability to deploy these instruments in a portable format.
Thermal convection has emerged as a promising alternative thermocycling approach that has the potential to overcome these limitations2-9. Convective flows are an everyday occurrence in a diverse array of settings ranging from the Earth's atmosphere, oceans, and interior, to decorative and colorful lava lamps. Fluid motion is initiated in the same way in each case: a buoyancy driven instability arises when a confined volume of fluid is subjected to a spatial temperature gradient. These same phenomena offer an attractive way to perform PCR thermocycling. By applying a static temperature gradient across an appropriately designed reactor geometry, a continuous circulatory flow can be established that will repeatedly transport PCR reagents through temperature zones associated with the denaturing, annealing, and extension stages of the reaction (Figure 1). Thermocycling can therefore be actuated in a pseudo-isothermal manner by simply holding two opposing surfaces at fixed temperatures, completely eliminating the need to repeatedly heat and cool the instrument.
One of the main challenges facing design of convective thermocyclers is the need to precisely control the spatial velocity and temperature distributions within the reactor to ensure that the reagents sequentially occupy the correct temperature zones for a sufficient period of time10,11. Here we describe results of our efforts to probe the full 3-D velocity and temperature distributions in microscale convective thermocyclers12. Unexpectedly, we have discovered a subset of complex flow trajectories that are highly favorable for PCR due to a synergistic combination of (1) continuous exchange among flow paths that provides an enhanced opportunity for reagents to sample the full range of optimal temperature profiles, and (2) increased time spent within the extension temperature zone the rate limiting step of PCR. Extremely rapid DNA amplification times (under 10 min) are achievable in reactors designed to generate these flows.

We describe a novel method to perform DNA replication via the polymerase chain reaction (PCR). Thermal convection is harnessed to continuously shuttle reagents between denaturing, annealing, and extension conditions by maintaining opposing surfaces of the reactor at constant temperature. This inherently simple design promises to make rapid PCR more accessible.

PCR Thermocycling السريع باستخدام الحراري الحرارية الميكروسكيل

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable ...

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

الفرز الفائق الإنتاجية وBiosensing مع نيون C. ايليجانس سلالات

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

الكشف عن تفاعلات البروتين في النبات باستخدام عبارة الإسفار ثنائي الجزيء المتوافقة التكملة (BiFC) نظام

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

Quantifying Mixing using Magnetic Resonance Imaging

Mixing is a unit operation that combines two or more components into a homogeneous mixture. This work involves mixing two viscous liquid streams using an in-line static mixer. The mixer is a split-and-recombine design that employs shear and extensional flow to increase the interfacial contact between the components. A prototype split-and-recombine (SAR) mixer was constructed by aligning a series of thin laser-cut Poly (methyl methacrylate) (PMMA) plates held in place in a PVC pipe. Mixing in this device is illustrated in the photograph in Fig. 1. Red dye was added to a portion of the test fluid and used as the minor component being mixed into the major (undyed) component. At the inlet of the mixer, the injected layer of tracer fluid is split into two layers as it flows through the mixing section. On each subsequent mixing section, the number of horizontal layers is duplicated. Ultimately, the single stream of dye is uniformly dispersed throughout the cross section of the device.
 Using a non-Newtonian test fluid of 0.2% Carbopol and a doped tracer fluid of similar composition, mixing in the unit is visualized using magnetic resonance imaging (MRI). MRI is a very powerful experimental probe of molecular chemical and physical environment as well as sample structure on the length scales from microns to centimeters. This sensitivity has resulted in broad application of these techniques to characterize physical, chemical and/or biological properties of materials ranging from humans to foods to porous media 1, 2. The equipment and conditions used here are suitable for imaging liquids containing substantial amounts of NMR mobile 1H such as ordinary water and organic liquids including oils. Traditionally MRI has utilized super conducting magnets which are not suitable for industrial environments and not portable within a laboratory (Fig. 2). Recent advances in magnet technology have permitted the construction of large volume industrially compatible magnets suitable for imaging process flows. Here, MRI provides spatially resolved component concentrations at different axial locations during the mixing process. This work documents real-time mixing of highly viscous fluids via distributive mixing with an application to personal care products.

Magnetic resonance imaging (MRI) provides a powerful tool to evaluate the effectiveness of process equipment during operation. We discuss the use of MRI to visualize mixing in a static mixer. The application is relevant to personal care products, but can be applied to a broad range of food, chemical, biomass and biological fluids.

خلط الكمي باستخدام التصوير بالرنين المغناطيسي

Mixing is a unit operation that combines two or more components into a homogeneous mixture.  This work involves mixing two viscous liquid streams using an ...

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community.

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation is a commonly employed method for introducing DNA into bacteria in a process known as transformation. Traditional protocols for the preparation of electrocompetent cells are time consuming and labor intensive. This article describes an alternate, rapid, and efficient method for the preparation of electrocompetent cells presently employed by some laboratories.

بروتوكول السريع لإعداد Electrocompetent<em&gt; كولاي</em&gt; و<em&gt; ضمة الكوليرا</em

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

سريعة وفعالة الزرد التنميط الجيني باستخدام PCR عالية الدقة مع تحليل نذوب

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

قراءات السائبة بسيطة الرقمية نوكليك حمض الكمي فحوصات

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents (HPHC)

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it is challenging to assess the contributions of individual constituents to its overall toxicity. Toxicological profiles of individual constituents as well as mixtures can be however established in vitro, by applying high through-put screening tools, which enable the profiling of Harmful and Potentially Harmful Constituents (HPHCs) of tobacco smoke, as defined by the U.S. Food and Drug Administration (FDA).2
For an initial assessment, an impedance-based instrument was used for a real-time, label-free assessment of the compound&#39;s toxicity. The instrument readout relies on cell adhesion, viability and morphology that all together provide an overview of the cell status. A dimensionless parameter, named cell index, is used for quantification. A set of different staining protocols was developed for a fluorescence imaging-based investigation and a HCS platform was used to gain more in-depth information on the kind of cytotoxicity elicited by each HPHC.
Of the 15 constituents tested, only five were selected for HCS-based analysis as they registered a computable LD50 (&#60; 20 mM). These included 1-aminonaphtalene, Arsenic (V), Chromium (VI), Crotonaldehyde and Phenol. Based on their effect in the HCS, 1-aminonaphtalene and Phenol could be identified to induce mitochondrial dysfunction, and, together with Chromium (VI) as genotoxic based on the increased histone H2AX phosphorylation. Crotonaldehyde was identified as an oxidative stress inducer and Arsenic as a stress kinase pathway activator.
This study demonstrates that a combination of impedance-based and HCS technologies provides a robust tool for in vitro assessment of CS constituents.

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents HPHC

The objective of the study was to assess the biological impact of 15 cigarette smoke constituents using a combination of an impedance-based real time cell analyzer and a high-content screening (HCS)-based platform for toxicological assessment in vitro. This study provides information on effective doses, toxicity and modes of action of the tested compounds.

عالية تحليل فحص المحتوى لتقييم الآثار السمية للمكونات الضارة والمؤذية يحتمل (HPHC)

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it ...