Dr Alexandre Mourot el yazması üzerine onun yorum için teşekkür. Bu eser Agence Nationale de la Recherche (ANR 2011 MALZ 003 01; gelen hibe tarafından desteklenmiştir ANR-15-CE16-0010 ve ANR-17-CE37-0010-03), BLG Fondation pour la Recherche sur Alzheimer bursu tarafından desteklenmektedir. IBPS (Paris, Fransa) hayvan tesisi teşekkür ediyoruz.

Yazarlar ifşa gerek yok.

Tek hücre multiplex RT-PCR electrophysiologically tanımlanan hücreleri530'dan fazla genlerin ifade aynı anda ve güvenilir bir şekilde yama-kelepçe yoklama sonra. Gen ifadesinin tek hücre düzeyinde analiz yüksek verimli PCR astar gerektirir. En sınırlayıcı adımlardan biri hücrenin içerik topluluğudur. Verimlilik hücre boyutu eşleştirme sırasında mümkün olduğu kadar büyük olmalıdır yama pipet ucu çapı göre değişir. Pipetler 1-2 µm açık uç çapı en nöronal tü...

Serebral korteks çok sayıda hücre tipleri çeşitli fizyolojik süreçlerinde yer oluşmaktadır. Kendi kimlik ve karakterizasyonu, özel işlevleriyle, anlayış için bir ön koşul kortikal hücre tip1 karakterize büyük morfolojik, fizyolojik ve moleküler çeşitliliği göz önüne alındığında çok zor olabilir ,2,3,4.
Tek hücreli multiplex RT-PCR yama-kelepçe ve RT-PCR teknikleri kombinasyonuna dayanmaktadır. Aynı anda electrophysiologically tanımlanan hücreleri530'dan fazla önceden tanımlanmış genlerin ifade sonda. Histochemical vahiy6,7,8,9, sonra kaydedilen hücrelerin karakterizasyonu morfolojik dahil edilmesi daha fazla kayıt pipet nöronal bir izleyici sağlar 10. onların fenotipik özellikleri5,9,10,11,12 çok değişkenli analize dayalı nöronal türleri sınıflandırılması için çok kullanışlı bir yöntemdir ,13,14. Tek hücre multiplex RT-PCR da astrocytes15,16,17gibi sigara nöronal hücrelerin karakterizasyonu için uygundur ve hemen hemen her beyin yapısı-18uygulanabilir, onlar bütün hücreli yapılandırmada kaydedilebilir varsayarak 19,20,21,22,23 ve hücre tipi.
Bu teknik çok hücresel kaynakları tanımlaması ve/veya iletim sistemleri7,8,15,16,20,21hedefleri için uygundur, özellikle belirli antikor eksik zaman 24,25,26,27,28,. Bu yama-kelepçe kayıtları görsel olarak tanımlanan hücreleri29kullanır ve böylece aynı zamanda hücreleri belirli hücresel ortamı8,15,16hedefleme sağlar. Beyin dokusu cytoarchitecture beyin dilimler halinde korunmuş olduğundan, Ayrıca bu yaklaşım da anatomik ilişkileri nöronal ve nöronal elements7,8 ile karakterize hücrelerinin çalışma sağlar , 18.
Bu tekniği hasat sitoplazma miktarı ve RT verimliliğini tarafından sınırlı olduğu için düşük kopya sayısı ifade mRNA tespiti zor olabilir. Yüksek üretilen iş pahalı sıralayıcılar ihtiyaçları ille RNaseq teknolojisine dayalı diğer yaklaşımlar tek hücreleri3,4,30,31, Bütün transcriptome analiz etmek için izin rağmen Her laboratuvar için kullanılabilir. Tek hücre multiplex RT-PCR tekniği son nokta PCR kullandığından, yalnızca yaygın olarak kullanılan thermocyclers gerektirir. Bu kolayca elektrofizyolojik set-up ile donatılmış laboratuarlarında geliştirilen ve pahalı ekipman gerektirmez. O, bir gün içinde bir kalitatif analiz genler önceden tanımlanmış bir dizi ifade sağlayabilir. Bu nedenle, bu yaklaşım hızlı bir şekilde tek hücreleri moleküler karakterizasyonu kolay bir erişim sunmaktadır.

<table><tbody><tr><td>MACAW v.2.0.5</td><td>NCBI</td><td></td><td>Multiple alignement for primer design</td></tr><tr><td>Dithiothreitol</td><td>VWR</td><td>443852A</td><td>RT</td></tr><tr><td>Random primers</td><td>Sigma-Aldrich (Merck)</td><td>11034731001</td><td>RT</td></tr><tr><td>dNTPs</td><td>GE Healthcare Life Sciences</td><td>28-4065-52</td><td>RT and PCR</td></tr><tr><td>RNasin Ribonuclease Inhibitors</td><td>Promega</td><td>N2511</td><td>RT</td></tr><tr><td>SuperScript II Reverse Transcriptase</td><td>Invitrogen</td><td>18064014</td><td>RT</td></tr><tr><td>Taq DNA Polymerase</td><td>Qiagen</td><td>201205</td><td>PCR</td></tr><tr><td>Mineral Oil</td><td>Sigma-Aldrich (Merck)</td><td>M5904-5ML</td><td>PCR</td></tr><tr><td>PCR primers</td><td>Sigma-Aldrich (Merck)</td><td></td><td>PCR / desalted and diluted at 200 &amp;micro;M</td></tr><tr><td>Tubes, 0.5 mL, flat cap</td><td>ThermoFisher Scientific</td><td>AB0350</td><td>RT and PCR</td></tr><tr><td>BT10 Series - 10 &amp;micro;L Filter Tip</td><td>Neptune Scientific</td><td>BT10</td><td>RT and PCR</td></tr><tr><td>BT20 Series - 20 &amp;micro;L Filter Tip</td><td>Neptune Scientific</td><td>BT20</td><td>RT and PCR</td></tr><tr><td>BT200 Series - 200 &amp;micro;L Filter Tip</td><td>Neptune Scientific</td><td>BT200</td><td>RT and PCR</td></tr><tr><td>BT1000 Series - 1000 &amp;micro;L Filter Tip</td><td>Neptune Scientific</td><td>BT1000.96</td><td>RT and PCR</td></tr><tr><td>DNA Thermal Cylcer</td><td>Perkin Elmer Cetus</td><td></td><td>PCR</td></tr><tr><td>Ethidium Bromide</td><td>Sigma-Aldrich (Merck)</td><td>E1510-10ML</td><td>Agarose gel electrophoresis</td></tr><tr><td>Tris-Borate-EDTA buffer</td><td>Sigma-Aldrich (Merck)</td><td>T4415-1L</td><td>Agarose gel electrophoresis</td></tr><tr><td>UltraPure Agarose</td><td>Life Technologies</td><td>16500-500</td><td>Agarose gel electrophoresis</td></tr><tr><td>&amp;Phi;X174 DNA-Hae III Digest</td><td>NEB (New England BioLabs)</td><td>N3026S</td><td>Agarose gel electrophoresis</td></tr><tr><td>EDA 290</td><td>Kodak</td><td></td><td>Agarose gel electrophoresis</td></tr><tr><td>Electrophoresis Power supply EPS 3500</td><td>Pharmacia Biotech</td><td></td><td>Agarose gel electrophoresis</td></tr><tr><td>Midi Horizontal Elecrophoresis Unit Model SHU13</td><td>Sigma-Aldrich (Merck)</td><td></td><td>Agarose gel electrophoresis</td></tr><tr><td>Smooth paper with satin appearance</td><td>Fisherbrand</td><td>1748B</td><td>Patch clamp internal solution</td></tr><tr><td>Potassium Hydroxyde</td><td>Sigma-Aldrich (Merck)</td><td>60377</td><td>Patch clamp internal solution</td></tr><tr><td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&amp;prime;,N&amp;prime;-tetraacetic acid</td><td>Sigma-Aldrich (Merck)</td><td>E3889</td><td>Patch clamp internal solution</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich (Merck)</td><td>H4034</td><td>Patch clamp internal solution</td></tr><tr><td>Potassium D-gluconate</td><td>Sigma-Aldrich (Merck)</td><td>G4500</td><td>Patch clamp internal solution</td></tr><tr><td>Magnesium chloride solution</td><td>Sigma-Aldrich (Merck)</td><td>M1028</td><td>Patch clamp internal solution</td></tr><tr><td>5500 Vapor Pressure Osmometer</td><td>Wescor</td><td></td><td>Patch clamp internal solution</td></tr><tr><td>Biocytin</td><td>Sigma-Aldrich (Merck)</td><td>B4261</td><td>Patch clamp internal solution</td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich (Merck)</td><td>S5016</td><td>Slice preparation</td></tr><tr><td>D-(+)-Glucose monohydrate</td><td>Sigma-Aldrich (Merck)</td><td>49159</td><td>Slice preparation</td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich (Merck)</td><td>S6191</td><td>Slice preparation</td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich (Merck)</td><td>60128</td><td>Slice preparation</td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich (Merck)</td><td>31437-M</td><td>Slice preparation</td></tr><tr><td>Sodium phosphate monobasic</td><td>Sigma-Aldrich (Merck)</td><td>S5011</td><td>Slice preparation</td></tr><tr><td>Magnesium chloride solution</td><td>Sigma-Aldrich (Merck)</td><td>63069</td><td>Slice preparation</td></tr><tr><td>Calcium chloride solution</td><td>Sigma-Aldrich (Merck)</td><td>21115</td><td>Slice preparation</td></tr><tr><td>Kynurenic acid</td><td>Sigma-Aldrich (Merck)</td><td>K3375</td><td>Slice preparation</td></tr><tr><td>Isoflurane</td><td>Piramal Healthcare UK</td><td></td><td>Slice preparation</td></tr><tr><td>VT 1000S</td><td>Leica Biosystems</td><td>14047235613</td><td>Slice preparation</td></tr><tr><td>Hydrogen peroxide solution</td><td>Sigma-Aldrich (Merck)</td><td>H1009</td><td>Patch Clamp set-up cleaning</td></tr><tr><td>Thin Wall Glass Capillaries with filament</td><td>World Precision Instruments</td><td>TW150F-4</td><td>Patch Clamp</td></tr><tr><td>PP-83</td><td>Narishige</td><td></td><td>Patch Clamp</td></tr><tr><td>Eppendorf Microloader</td><td>Eppendorf</td><td>5242956003</td><td>Patch Clamp</td></tr><tr><td>BX51WI Upright microscope</td><td>Olympus</td><td></td><td>Patch Clamp</td></tr><tr><td>XC-ST70/CE CCD B/W VIDEO CAMERA</td><td>Sony</td><td></td><td>Patch Clamp</td></tr><tr><td>Axopatch 200B Amplifier</td><td>Molecular Devices</td><td></td><td>Patch Clamp</td></tr><tr><td>Digidata 1440</td><td>Molecular Devices</td><td></td><td>Patch Clamp</td></tr><tr><td>pCLAMP 10 software suite</td><td>Molecular Devices</td><td></td><td>Patch Clamp</td></tr><tr><td>10 mL syringe</td><td>Terumo</td><td>SS-10ES</td><td>Expelling</td></tr><tr><td>E Series with Straight Body (Holder)</td><td>Phymep</td><td>64-0997</td><td>Expelling</td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma-Aldrich (Merck)</td><td>S7907</td><td>Histochemical revelation</td></tr><tr><td>Sodium phosphate monobasic</td><td>Sigma-Aldrich (Merck)</td><td>S8282</td><td>Histochemical revelation</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich (Merck)</td><td>P6148</td><td>Histochemical revelation</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich (Merck)</td><td>X100</td><td>Histochemical revelation</td></tr><tr><td>Gelatin from cold water fish skin</td><td>Sigma-Aldrich (Merck)</td><td>G7041</td><td>Histochemical revelation</td></tr><tr><td>Streptavidin, Alexa Fluor 488 conjugate</td><td>ThermoFisher Scientific</td><td>S11223</td><td>Histochemical revelation</td></tr><tr><td>24-well plate</td><td>Greiner Bio-One</td><td>662160</td><td>Histochemical revelation</td></tr></tbody></table>

single cell multiplex reverse transcription polymerase chain reaction after patch-clamp

Serebral korteks çeşitli morfolojik, fizyolojik ve moleküler özellikler sergileyen çok sayıda hücre türleri oluşur. Bu hücre türleri, özel işlevleriyle çalışmak için Önkoşullar malzemelerin kolay tanımlanması ve bu çeşitliliği engellemektedir. Bu makalede, sonra yama-kelepçe dilimler halinde kayıt aynı anda onlarca genlerin ifade tek bir hücreye algılamak için sağlar multiplex tek hücre Ters transkripsiyon polimeraz zincir reaksiyonu (RT-PCR) protokolünü açıklar. Bu basit yöntem ile morfolojik karakterizasyonu uygulanabilir ve kan damarlarının çevresinde gibi çeşitli hücre tiplerinin ve belirli hücresel çevreleri, fenotipik özellikleri belirlemek için yaygın olarak uygulanabilir. Bu iletişim kuralı bir hücre hasat ve ters yama-klemp tekniği ile uyarlamak sitoplazmik içeriğini kaydetmek için ve niteliksel algılamaya genler tarafından önceden tanımlanmış bir dizi ifade multiplex PCR ilkedir. PCR astar ve hücre içi yama-kelepçe çözüm RT-PCR ile uyumlu dikkatli bir tasarım gerektirir. Seçici ve güvenilir transkript sağlamak için algılama, bu teknik aynı zamanda sitoplazma amplifikasyon merdiven-e doğru hasat uygun denetimlerinin gerektirir. Burada tartışılan önlemler kesinlikle takip edilmesi gereken rağmen hemen hemen her elektrofizyolojik laboratuvar multiplex tek hücre RT-PCR tekniği kullanabilirsiniz.

Multiplex RT-PCR temsilcisi doğrulamasına Şekil 3' te gösterilmiştir. Protokol aynı anda 12 farklı genler ifade soruşturma için tasarlanmıştır. Veziküler glutamat ışınlama vGluT1 glutamatergic nöronlar42için pozitif kontrol olarak alınmıştır. Enzimler (GAD65 ve GAD 67), peptit Y (NPY) ve Somatostatin (SOM) sentezleme GABA GABAergic interneurons3,5,</s...

Watch this Scientific Journal Video about Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp at JoVE.com

Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp

Tek hücre Multiplex Ters transkripsiyon polimeraz zincir tepkimesi sonra yama-kelepçe

Bu iletişim kuralı kritik adımlar ve yama-kelepçe sonra tek hücre multiplex Ters transkripsiyon polimeraz zincir reaksiyonu gerçekleştirmek için gereken önlemleri açıklar. Bu teknik ifade profil genler tarafından yama-kelepçe kayıtları ile karakterize bir tek hücreden önceden belirlenmiş bir dizi analiz etmek için basit ve etkili bir yöntemdir.

The cerebral cortex is composed of numerous cell types exhibiting various morphological, physiological, and molecular features. This diversity hampers ...

single-cell-multiplex-reverse-transcription-polymerase-chain-reaction

Research

JoVE Journal

Neuroscience

9.7K Görüntüleme.  Sorbonne Université. Bu iletişim kuralı kritik adımlar ve yama-kelepçe sonra tek hücre multiplex Ters transkripsiyon polimeraz zincir reaksiyonu gerçekleştirmek için gereken önlemleri açıklar. Bu teknik ifade profil genler tarafından yama-kelepçe kayıtları ile karakterize bir tek hücreden önceden belirlenmiş bir dizi analiz etmek için basit ve etkili bir yöntemdir.

Video: Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp

Profiling Voltage-gated Potassium Channel mRNA Expression in Nigral Neurons using Single-cell RT-PCR Techniques

In mammalian central nervous system, different types of neurons with diverse molecular and functional characteristics are intermingled with each other, difficult to separate and also not easily identified by their morphology. Thus, it is often difficult to analyze gene expression in a specific neuron type. Here we document a procedure that combines whole-cell patch clamp recording techniques with single-cell reverse transcription polymerase chain reaction (scRT-PCR) to profile mRNA expression in different types of neurons in the substantial nigra. Electrophysiological techniques are first used to record the neurophysiological and functional properties of individual neurons. Then, the cytoplasm of single electrophysiologically characterized nigral neurons is aspirated and subjected to scRT-PCR analysis to obtain mRNA expression profiles for neurotransmitter synthesis enzymes, receptors, and ion channels. The high selectivity and sensitivity make this method particularly useful when immunohistochemistry can not be used due to a lack of suitable antibody or low expression level of the protein. This method is also applicable to neurons in other brain areas.

Neurons are first characterized electrophysiologically. Then the cytoplasm from the recorded neuron is aspirated and subjected to reverse transcription-PCR analysis to detect the expression of mRNAs for neurotransmitter synthesis enzymes, ion channels, and receptors.

Voltaj-kapılı potasyum Kanal mRNA gösterilmeden Tek hücreli RT-PCR teknikleri kullanarak Nigral Nöronlar

In mammalian central nervous system, different types of neurons with diverse molecular and functional characteristics are intermingled with each other, ...

Nörobilim

Single-cell Profiling of Developing and Mature Retinal Neurons

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and gene expression programs for extremely rare cell subsets has been a challenge using standard whole-tissue approaches. Gene expression profiling of individual cells allows for unprecedented access to cell types that comprise only a small percentage of the total tissue1-7. In addition, this technique can be used to examine the gene expression programs that are transiently expressed in small numbers of cells during dynamic developmental transitions8.
This issue of cellular diversity arises repeatedly in the central nervous system (CNS) where neuronal connections can occur between quite diverse cells9. The exact number of distinct cell types is not precisely known, but it has been estimated that there may be as many as 1000 different types in the cortex itself10. The function(s) of complex neural circuits may rely on some of the rare neuronal types and the genes they express. By identifying new markers and helping to molecularly classify different neurons, the single-cell approach is particularly useful in the analysis of cell types in the nervous system. It may also help to elucidate mechanisms of neural development by identifying differentially expressed genes and gene pathways during early stages of neuronal progenitor development.
As a simple, easily accessed tissue with considerable neuronal diversity, the vertebrate retina is an excellent model system for studying the processes of cellular development, neuronal differentiation and neuronal diversification. However, as in other parts of the CNS, this cellular diversity can present a problem for determining the genetic pathways that drive retinal progenitors to adopt a specific cell fate, especially given that rod photoreceptors make up the majority of the total retinal cell population11. Here we report a method for the identification of the transcripts expressed in single retinal cells (Figure 1). The single-cell profiling technique allows for the assessment of the amount of heterogeneity present within different cellular populations of the retina2,4,5,12. In addition, this method has revealed a host of new candidate genes that may play role(s) in the cell fate decision-making processes that occur in subsets of retinal progenitor cells8. With some simple adjustments to the protocol, this technique can be utilized for many different tissues and cell types.

A method for the isolation of single retinal cells and subsequent amplification of their cDNAs is described. Single-cell transcriptomics reveals the degree of cellular heterogeneity present in a tissue and uncovers new marker genes for rare cell populations. The accompanying protocol can be adjusted to suit many different cell types.

Gelişen ve Olgun Retina Nöronlar Tek hücreli Profilleme

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and ...

A Computer-assisted Multi-electrode Patch-clamp System

The patch-clamp technique is today the most well-established method for recording electrical activity from individual neurons or their subcellular compartments. Nevertheless, achieving stable recordings, even from individual cells, remains a time-consuming procedure of considerable complexity. Automation of many steps in conjunction with efficient information display can greatly assist experimentalists in performing a larger number of recordings with greater reliability and in less time. In order to achieve large-scale recordings we concluded the most efficient approach is not to fully automatize the process but to simplify the experimental steps and reduce the chances of human error while efficiently incorporating the experimenter's experience and visual feedback. With these goals in mind we developed a computer-assisted system which centralizes all the controls necessary for a multi-electrode patch-clamp experiment in a single interface, a commercially available wireless gamepad, while displaying experiment related information and guidance cues on the computer screen. Here we describe the different components of the system which allowed us to reduce the time required for achieving the recording configuration and substantially increase the chances of successfully recording large numbers of neurons simultaneously.

Multi-electrode patch-clamp recordings constitute a complex task. Here we show how, by automating of many of the experimental steps, it is possible to accelerate the process leading to qualitative improvement in performance and number of recordings.

Bilgisayar destekli Çoklu elektrot Patch-kelepçe Sistemi

The patch-clamp technique is today the most  well-established method for recording electrical activity from individual  neurons or their subcellular ...

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 ...

Biyoloji

Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) is a ubiquitous research strategy. HEK-293T cells must be plated onto glass coverslips at low enough density so that they are not in contact with each other in order to allow for electrophysiological recording without confounding effects due to contact with adjacent cells. Transfected channels must also express with high efficiency at the plasma membrane for whole-cell patch clamp recording of detectable currents above noise levels. Heterologous ion channels often require long incubation periods at 28&deg;C after transfection in order to achieve adequate membrane expression, but there are increasing losses of cell-coverslip adhesion and membrane stability at this temperature. To circumvent this problem, we developed an optimized strategy to transfect and plate HEK-293T cells. This method requires that cells be transfected at a relatively high confluency, and incubated at 28&deg;C for varying incubation periods post-transfection to allow for adequate ion channel protein expression. Transfected cells are then plated onto glass coverslips and incubated at 37&deg;C for several hours, which allows for rigid cell attachment to the coverslips and membrane restabilization. Cells can be recorded shortly after plating, or can be transferred to 28&deg;C for further incubation. We find that the initial incubation at 28&deg;C, after transfection but before plating, is key for the efficient expression of heterologous ion channels that normally do not express well at the plasma membrane. Positively transfected, cultured cells are identified by co-expressed eGFP or eGFP expressed from a bicistronic vector (e.g. pIRES2-EGFP) containing the recombinant ion channel cDNA just upstream of an internal ribosome entry site and an eGFP coding sequence. Whole-cell patch clamp recording requires specialized equipment, plus the crafting of polished recording electrodes and L-shaped ground electrodes from borosilicate glass. Drug delivery to study the pharmacology of ion channels can be achieved by directly micropipetting drugs into the recording dish, or by using microperfusion or gravity flow systems that produce uninterrupted streams of drug solution over recorded cells.

Reliable method for highly efficient in vitro expression and subsequent electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T).

İfade ve HEK-293T Hücreler Rekombinant Gerilim Gated İyon Kanalları Elektrofizyolojik Kayıt için optimize edilmiş Transfeksiyon Strateji

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) ...

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate electrical processes in target cells. In this context, ion selectivity of a channelrhodopsin is of particular importance. This article describes the investigation of chloride selectivity for a recently identified anion-conducting channelrhodopsin of Proteomonas sulcata via electrophysiological patch-clamp recordings on HEK293 cells. The experimental procedure for measuring light-gated photocurrents demands a fast switchable &#8211; ideally monochromatic &#8211; light source coupled into the microscope of an otherwise conventional patch-clamp setup. Preparative procedures prior to the experiment are outlined involving preparation of buffered solutions, considerations on liquid junction potentials, seeding and transfection of cells, and pulling of patch pipettes. The actual recording of current-voltage relations to determine the reversal potentials for different chloride concentrations takes place 24 h to 48 h after transfection. Finally, electrophysiological data are analyzed with respect to theoretical considerations of chloride conduction.

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.

Channelrhodopsins&#39;teki İyon Seçiciliğinin Elektrofizyolojik Olarak Belirlenmesi için Tam Hücreli Yama Kelepçesi Kayıtları

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

Yetişkin Fare kardiyomiyositlerde Tek Hücre transkripsiyonel Profilleme

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

Tek kanallı bir hücre-bağlı Patch-clamp kayıt

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Technical Applications of Microelectrode Array and Patch Clamp Recordings on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Drug-induced cardiotoxicity is the leading cause of drug attrition and withdrawal from the market. Therefore, using appropriate preclinical cardiac safety assessment models is a critical step during drug development. Currently, cardiac safety assessment is still highly dependent on animal studies. However, animal models are plagued by poor translational specificity to humans due to species-specific differences, particularly in terms of cardiac electrophysiological characteristics. Thus, there is an urgent need to develop a reliable, efficient, and human-based model for preclinical cardiac safety assessment. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as an invaluable in vitro model for drug-induced cardiotoxicity screening and disease modeling. hiPSC-CMs can be obtained from individuals with diverse genetic backgrounds and various diseased conditions, making them an ideal surrogate to assess drug-induced cardiotoxicity individually. Therefore, methodologies to comprehensively investigate the functional characteristics of hiPSC-CMs need to be established. In this protocol, we detail various functional assays that can be assessed on hiPSC-CMs, including the measurement of contractility, field potential, action potential, and calcium handling. Overall, the incorporation of hiPSC-CMs into preclinical cardiac safety assessment has the potential to revolutionize drug development.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a promising in vitro model for drug-induced cardiotoxicity screening and disease modeling. Here, we detail a protocol for measuring the contractility and electrophysiology of hiPSC-CMs.

İnsan Kaynaklı Pluripotent Kök Hücre Kaynaklı Kardiyomiyositler Üzerindeki Mikroelektrot Dizisi ve Yama Kelepçesi Kayıtlarının Teknik Uygulamaları

Drug-induced cardiotoxicity is the leading cause of drug attrition and withdrawal from the market. Therefore, using appropriate preclinical cardiac safety ...

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Establishing a Whole-Cell Configuration through the Patch Clamp Method

Source: Segev, A. et al., <a href="https://app.jove.com/v/54024">Whole-cell Patch-clamp Recordings in Brain Slices.</a>&#160;J. Vis. Exp.&#160;(2016)
This video demonstrates the whole-cell patch clamp technique in a mouse brain slice. This technique establishes a stable connection between micropipette and neuron membrane, which is further disrupted to establish a whole-cell configuration to study the neuron&#39;s health and physiological responses.

Yama Kelepçesi Yöntemi ile Tüm Hücre Konfigürasyonunun Oluşturulması

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Slice ...