We thank Lowell Cochran for expert videography assistance. The financial assistance of the Canadian Institutes of Health Research (CIHR), the Canadian Breast Cancer Foundation (CBCF, Ontario Chapter), the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canadian Breast Cancer Research Alliance, the Ontario Centers of Excellence, the Breast Cancer Action Kingston and the Clare Nelson bequest fund through grants to LR is gratefully acknowledged. SG was the recipient of an NSERC studentship. MG was supported by a postdoctoral fellowship from the US Army Breast Cancer Program, the Ministry of Research and Innovation of the Province of Ontario and the Advisory Research Committee of Queen&#8217;s University.

1. Elektroporasyon Chambers Hücreleri Kaplama <ol><li> Laminer akış kaputu, her zamanki gibi steril tekniği kullanarak hücreleri trypsinize. Not: Bu, camın üzerine hücrelerin yayılmasını engelleyebilir, çünkü santrifüjle adherens ve boşluk bağlantıları bu nedenle oluşumunu tripsin tüm izlerini ortadan kaldırmak için çok önemlidir. </li><li> Pipet 1, 37 o C&#39;de in situ elektroporatörü içine (Şekil 1) ve yerine, CO2 inkübatör ile donatılmış steril elektroporasyon odalarında ml hücre süspansiyonu. Not: Hücre yapışması, fibronektin, kolajen, poli-lisin ya da hücre ve doku yapıştırıcı (Malzemeler Tabloya bakınız) üzerine kaplanarak artırılabilir. </li><li> Hücreler konfluent tabakası oluşturdular onlar GJIC inceleme için hazırdır. </li></ol> 2. Elektroporasyon Prosedürü GJIC muayene için elektroporasyon, bir laminer akış kaputu dışında yapılabiliralır bu yana sadece bir kaç dakika. Daha uzun inkubasyon süreleri belirli bir deney için gerekli olduğu durumda, o zaman bir laminer akış başlığı içinde tamamen yapılabilir. Tüm durumlarda, hücrelerin büyüdüğü odacıklar steril olması gerekmektedir. <ol><li> 5 mg / ml, Lucifer sarı bir çözelti hazırlayın: 2 mi Kalsiyum içermeyen büyüme ortamında (elektroporatörü ile birlikte verilme), 10 mg LY eritin. 4 o C&#39;deki solüsyonu ve mağaza filtre-sterilize, uzun inkübasyon sürelerine ihtiyaç duyar deneyler için </li><li> Büyüme ortamı aspire ve tek tabaka ya da sıfırdan hücreleri kurutmak için dikkatli olmak, kalsiyum-barındırmayan ortam maddesi ile hücreleri yıkayın. Hücreler kurutulur varsa genellikle koyu çekirdekleri ve elektroporasyon olmadan LY al. Etkisi daha hava akımı (Şekil 4F) maruz slayt ortasında daha belirgindir. </li><li> Bir Eppendorf pipet ile, bölme, B kenarında, hücreler (bütün odası için 400 ul), boya çözeltisinin pipetleDikkatli eing hücre tabakasını dokunmamaya. </li><li> Elektroporatörü ile birlikte tutucu odasına yerleştirin ve uygun kuvvette (Tartışma) bir darbe uygulamak. </li><li> Dikkatle LY çözümün bazı aspire. Bu deneyler için daha az önemli bir ikinci kez tekrar edilebilir. </li><li> ,% 10 serum ihtiva eden diyaliz kalsiyum içermeyen DMEM ekleyin. </li><li> Boşluk bağlantıları ile boya transferini sağlamak için inkübatör 3-5 dakika için kuluçkalayın. Not: Bu noktada, diyaliz serum dahil açıklıklarının kapanmasına yardımcı olur. </li><li> Canlı hücre gözlem için Kalsiyum içermeyen DMEM ile tüzel kişiliği olmayan boya yıkayın. Seçenek olarak ise, hücreler çukuruna% 4 formaldehit ilave edilmek suretiyle bu aşamada tespit edilebilir, daha sonra PBS (fosfat-tamponlu tuzlu su) ile yıkanmıştır. Tüm durumlarda yıkama tüm arka planı kaldırmak gerekir. NOT: Ön deneyler gerilimi çok düşükse, uygun koşulları belirlemek amacıyla, o hücrelerin incubato kurtarmak izin mümkündürBir kaç dakika boyunca r ve slaytlar maliyeti kurtarmak için, aynı Sürgü ikinci kez electroporate. </li></ol> 3. Mikroskobik İnceleme <ol><li> Kullanılan boya için uygun bir filtre ile donatılmış bir ters çevrilmiş bir mikroskop kullanarak, floresan aydınlatma altında hücreleri dikkate alınmalıdır. Sarı Lucifer, uyarma 423nm, emisyon 555nm, Nikon IX70 mikroskop için WBV filtre. </li><li> Faz kontrast altında hücre morfolojisini incelemek için, sıvı ile üst doldurduktan sonra, iyi plastik üstüne bir cam kapak-slip koyarak menisküs etkilerini ortadan kaldırmak. Daha iyi fotoğraflar için, iyi slayt ayrılabilir, ve lamel çerçeveye yerleştirilir. Canlı hücreler için bu büyüme ortamı ile doldurulması gerekir ise sabit hücreler için de, mikroskopik gözlem için gliserol ile doldurulabilir. NOT: Hücreler bir boya (yukarıdaki aşama 2.7) transferi aşağıdaki formaldehitle sabitlendi, eğer yıkama ve görüntüleme daha kolaydır. Hücreler ise hiçbirT floresan sabit hücrelerin floresans birkaç saat boyunca muhafaza edilir getirirken LY hücre tipi, gerilim ve miktarına bağlı olarak, yaklaşık 60 dakika içinde hücrelerden elimine edilir, sabit. Bununla birlikte, floresan kaybolur ya da sabit hücrelerde, O / N inkübasyondan sonra dağılır. Bu nedenle, fotoğraf elektroporasyon (Şekil 2) kısa bir süre sonra alınmalıdır. </li></ol> Hücreler arası İletişimin 4. Kantifikasyonu <ol><li> Floresans ve faz kontrast (Şekil 3) altında, bir 20x objektif ile hücrelerin fotoğraf. </li><li> Olmayan elektroporasyona alanı (Şekil 2, ok, elektroporasyon kenar) ile sınırında elektropore hücreleri belirlemek ve bir yıldız ile işaretleyin. </li><li> Boya boşluk bağlantıları aracılığıyla transfer olan-olmayan elektroporasyona tarafında, floresanlama hücreleri belirlemek ve bir işaret ile (Şekil 3A ve 3B). </li><li> Toplam sayısına bölünkenar (Şekil 3A ve 3B) boyunca elektroporate edilmiş hücrelerde sayısına göre olmayan elektroporasyona alanda hücreleri fluoresan. NOT: en az 200 bitişik Elektroporasyona çevresindeki hücrelerden Transfer, her bir deney için hesaplanır. Elde edilen sayı GJIC değerdir. </li></ol>

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The corresponding author is the inventor in a patent held by Queen&#8217;s University, which has been licensed to Cell Projects Inc. The company or the University had no influence upon the contents of the paper.

Protokolünde kritik adımlar Elektroporasyona malzeme Elektropore edilecekse Malzemenin saflığı çok önemlidir. Hücreler, çok düz ise, izleme boyanın daha yüksek konsantrasyonlarda (20 mg LY / ml kadar) ve bu gibi durumlarda kullanılmalıdır, saflık: daha da önemlisi, daha küresel bir şekle sahip 1 hücrelerinde daha uzundur. LY Bunun yanı sıra, diğer boyalar ya da nonpermeant moleküllerin büyük bir çeşitlilik, örneğin başka bir con...

Bir hücre için elektrik akımının uygulanması işlemi olarak adlandırılan bir elektroporasyon ile, hücre zarı üzerindeki gözeneklerin oluşmasına neden olur. Gözenekler zardan nonpermeant çeşitli moleküllerin geçişine izin verir. Elektrik alanı meydana getirilen gözenekler, çok küçük ve hücre fizyolojisinin çok az rahatsızlık ile, hızlı bir şekilde yeniden kapatmak böylece, hassas bir şekilde kontrol edilebilir. İlginç bir şekilde, yapışkan hücreler, süspansiyon içinde elektropore edilecekse koparılmadan, büyümek yüzeyi üzerinde iletken ve şeffaf indiyum-kalay oksit (İTO) ile kaplanmış ve in situ elektroporasyona bir cam slayt üzerinde büyütülebilir. Hücreler, bu yüzeyde çok iyi büyüyebilir, ve bağlı ve uzatıldıkça detaylı mikroskobik gözlem mümkündür. Bu tekniği kullanarak, küçük moleküller nonpermeant anında sunulan ve comp aktivasyonu üzerinde çalışmalar için özellikle uygun hale getirir, bu teknik, hücrelerin esas olarak% 100, içine edilebilir(1 gözden) bir reseptörünün ligand stimülasyon sonra yolun onents. Elektroporasyon DNA&#39;nın katılımı (buna electrotransfection) çoğunlukla kullanılmaktadır. Bununla birlikte, in situ elektroporasyon transkripsiyon faktörü bağlanma, veya siRNA 3,5 inhibe etmek için örneğin antisens RNA gibi çift iplikli DNA Decoy-Oligonükleotidleri gibi peptidler 2-4, oligonükleotidler gibi moleküller, büyük bir çeşitlilik sokulması için değerli olabilir, 6, radyoaktif nükleotidleri, 7-10, proteinler 11 12 ya da ön-ilaçları 13. Elektroporasyondan sonra hücreler, biyokimyasal analizler için lize veya sabit ve antikorlar ile boyanmış olabilir. Hücreler, iletken kaplamanın kenarı boyunca büyüdükçe hücre büyümesi, iki yüzeyin yükseklik farkından rahatsız etmeyecek şekilde iletken İTO kaplama, 800-1,000Å çok incedir. Bu durum şu avantaja sahiptir olmayan electropbu metne dahil edilmiştir hücreler kontrol olarak hizmet etmek üzere, elektroporasyona olanlar ile yan yana yetiştirilebilir. Video tarif edildiği gibi aynı yaklaşım, boşluk junctional, hücreler arası iletişim (GJIC) &#39;nin incelenmesi için de kullanılabilir. Gap kavşaklar komşu hücrelerin 14 iç bağlayan kanallardır. Src gibi onkogenler GJIC 15,16 bastırırken boşluk birleşim, hücreler arası iletişim, tümör oluşumu ve metastaz bakımından önemli bir rol oynar. Lucifer sarının (LY) çoğu zaman mikro-enjeksiyon ile kültürlenmiş hücreler içine dahil edilebilmekte ya da kazıma-yükleme 17 ve komşu hücrelere boyanın difüzyonu mikroskopik floresan aydınlatma altında görülmektedir olduğu gibi GJIC bir floresan boya incelemek. Bu teknikler ancak kaçınılmaz hücre hasarına neden olur. Şimdi hücreleri kısmen İTO 18 ile kaplanmış bir cam slayt üzerinde yetiştirilen bir tekniği açıklar. Bir elektrik atma LY (ya da diğer boyalar) varlığında ca uygulanmıştırbitişik olmayan elektroporate edilmiş hücrelerde, boyanın göçü, mikroskopik floresan aydınlatma ile gözlenirken, sürgünün iletken kısmına üzerinde büyüyen hücrelerin içine nüfuziyetini kullanılmıştır. Tek tabaka ayırmak için temayül rahatsız duyarlı hücreler önlemek için, bir elektrot gerektirmeyen tasarlanmış bir montaj elektrik akımı 19 uygulamak için hücrelerin üzerine yerleştirilmesini mümkün kılar. Serum uygulandıktan 12 uygulanarak G1 fazında uzunluğuna bir etkisinin olmaması ile gösterildiği gibi bu yaklaşım fos düzeylerindeki artış, hücre metabolizması için, belirgin bir bozulma olmadan hücrelerin çok sayıda GJIC ölçmek için olanağı sunar protoonkojenden protein (Raptis, yayınlanmamış) veya hücresel stres, p38 domuz veya JNK / SAPK&#39;ın kinaz 1 ile ilişkili iki kinazlardır. Onkojen ifadesi, transformasyon ve GJ düzeyleri arasındaki bağlantının incelenmesi mümkün hale Bu yaklaşımIC 18, hem de akciğer tümör örneklerinde 20-23 taze kültürlenmiş hücreler de dahil olmak üzere hücre tiplerinin, çeşitli içinde GJIC üzerine Src ve STAT3 etkisi. Buna ek olarak, alt-tabakaya bağlanma hücresel bu aşamada 19,24 azaltılır, ancak başarılı adipositik farklılaşması üzerine boşluk bağlantı kapağın gösterilmesi için kullanılan edilmiş bir üst elektrot yoksun tarif etmiş olup in situ elektroporasyon.

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			<td height="21" style="height:21px;width:179px;">DMEM</td>
			<td style="width:428px;">Cellgro http://www.cellgro.com/</td>
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			<td height="21" style="height:21px;width:179px;">DMEM without Calcium</td>
			<td style="width:428px;">Hyclone (Thermo scientific: http://www.thermoscientific.com)</td>
			<td style="width:119px;">SH30319-01</td>
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			<td height="21" style="height:21px;width:179px;">Donor Calf Serum&#160;</td>
			<td style="width:428px;">PAA: http://www.paa.com/</td>
			<td style="width:119px;">Cat.# B15-008</td>
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			<td height="21" style="height:21px;width:179px;">Fetal Bovine Serum</td>
			<td style="width:428px;">PAA: http://www.paa.com/</td>
			<td style="width:119px;">Cat.# A15-751</td>
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			<td style="width:428px;">Cell Projects Ltd UK: http://www.cellprojects.com/</td>
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			<td>4-wells</td>
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			<td style="width:428px;">BD Biosciences</td>
			<td style="width:119px;">354240</td>
			<td>Cell and tissue adhesive</td>
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			<td style="width:428px;">Sigma Aldrich</td>
			<td style="width:119px;">F1141</td>
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			<td height="21" style="height:21px;width:179px;">Collagen</td>
			<td style="width:428px;">BD Biosciences</td>
			<td style="width:119px;">354236</td>
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			<td style="width:428px;">Sigma Aldrich</td>
			<td style="width:119px;">P8920</td>
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a functional assay for gap junctional examination; electroporation of adherent cells on indium-tin oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.&#160; Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive&#160;glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.

Şekil 2, tespit ve yıkama sonrası floresan (Panel A ve B) kapsamında LY ile elektropore edildi ve fotoğraflandı sıçan karaciğer epitel hücreleri T51B 22, ya da faz-kontrast (C) aydınlatma göstermektedir. Panel A, electroporated alanın kenar kırmızı olarak işaretlenmiştir. Kırmızı çizginin sağında flüoresans gradyanı boşluk bağlantıları yoluyla transferini ifade eder. Boya transfer olan hücreler, bir nokta ile işaretlenmiş ise, elektroporasyona alanının kenar...

Watch this Scientific Journal Video about A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide at JoVE.com

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

Gap Kavşak Sınavı Fonksiyonel Testi; İndiyum-Kalay Oksit Yapışık hücrelerin elektroporasyonu

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. ...

a-functional-assay-for-gap-junctional-examination-electroporation

Research

JoVE Journal

Biology

9.8K Görüntüleme.  Queen's University. This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

Video: A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

Patterning Cells on Optically Transparent Indium Tin Oxide Electrodes

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular constructs serving as in vitro mimics of native tissues.  In this study, photolithography and wet etching techniques were used to fabricate individually addressable indium tin oxide (ITO) electrodes on glass substrates.  The glass substrates containing ITO microelectrodes were modified with poly(ethylene glycol) (PEG) silane to make them protein and cell resistive.  Presence of insulating PEG molecules on the electrode surface was verified by cyclic voltammetry employing potassium ferricyanide as a redox reporter molecule.  Importantly, the application of reductive potential caused desorption of the PEG layer, resulting in regeneration of the conductive electrode surface and appearance of typical ferricyanide redox peaks.    Application of reductive potential also corresponded to switching of ITO electrode properties from cell non-adhesive to cell-adhesive. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.  Micropatterning of several cell types was demonstrated on these substrates.   In the future, the control of the biointerfacial properties afforded by this method will allow to engineer cellular microenvironments through the assembly of three or more cell types into a precise geometric configuration on an optically transparent substrate.

Non-fouling PEG silane monolayer was desorbed from individually addressable ITO electrodes on glass by application of a reductive potential. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.

Optik Şeffaf indiyum kalay oksit Elektrotlar Desenlendirme Hücreler

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular ...

Biyoloji

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

Demir Oksit hESC ve hMSCs Etiketleme MR Görüntüleme in vivo Takip Non-Invasive Nanopartiküller

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

Fare Retina Ganglion Hücreleri Gen İfadesi utero ve ex vivo Elektroporasyon

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

Yapışkan ve Sigara yapışık Memeli Hücre yerinde Subselüler Fraksiyonu

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

İyon Kanalları ve Fonksiyonel Analiz Mutagenez Heterologously Memeli Hücreleri cinsinden ifade edilir

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

Clonogenic Testi: yapışkan Hücreler

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3&#039;UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

3&#39;UTR muhabiri oluşturur ve bir microRNA kullanarak LightSwitch Lusiferaz Test Sistemi ile İnsan microRNA belirlenmesi Hedefler yapışık hücrelere Mimik

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific shapes. Drosophila tracheal terminal cells have become a powerful genetic model to identify and elucidate the roles of genes required for generating cellular morphologies. Terminal cells are a component of a branched tubular network, the tracheal system that functions to supply oxygen to internal tissues. Terminal cells are an excellent model for investigating questions of cell shape as they possess two distinct cellular architectures. First, terminal cells have an elaborate branched morphology, similar to complex neurons; second, terminal cell branches are formed as thin tubes and contain a membrane-bound intracellular lumen. Quantitative analysis of terminal cell branch number, branch organization and individual branch shape, can be used to provide information about the role of specific genetic mechanisms in the making of a branched cell. Analysis of tube formation in these cells can reveal conserved mechanisms of tubulogenesis common to other tubular networks, such as the vertebrate vasculature. Here we describe techniques that can be used to rapidly fix, image, and analyze both branching patterns and tube formation in terminal cells within Drosophila larvae. These techniques can be used to analyze terminal cells in wild-type and mutant animals, or genetic mosaics. Because of the high efficiency of this protocol, it is also well suited for genetic, RNAi-based, or drug screens in the Drosophila tracheal system.

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Here, we present a method for light microscopy analysis of tracheal terminal cells in Drosophila larvae. This method allows for quick examination of branch and lumen morphology in whole animals and would be useful for analysis of individual mutants or screens for mutations affecting terminal cell development.

İncelenmesi<em&gt; Drosophila</emIşık Mikroskopi tarafından&gt; Larva Trakeal Terminal Hücreleri

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Ekstraselüler Asit Üretim Ölçüm ve Analiz Glikolitik Hızı belirleme

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Zebra balığı Nephrotoxin Mikroenjeksiyon Akut Böbrek Yaralanması Model

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...