アルコール乱用の国民の協会からとPAのプリ博士NIH NRSAフェローシップ（F31AA017042）、私たちの活動は、神経科学（PAS）、国立薬物乱用研究所（PAS R01 DA019022）のためのマックナイト基金基金からの財政支援によって可能となったとアルコール依存症。 GIRK2 - L257W変異体は、博士プラフルラAryalによって提供されていました。 親切にライカインスツルメンツが提供する蛍光顕微鏡とスポンサーシップ。

<ol>
	<li>Aryal, P., Dvir, H., Choe, S., Slesinger, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+discrete+alcohol+pocket+involved+in+GIRK+channel+activation.">A discrete alcohol pocket involved in GIRK channel activation.</a> Nature Neurosci. 12, 988-995 (2009).</li><li>Panchenko, V. A., Glasser, C. R., Mayer, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+similarities+between+glutamate+receptor+channels+and+K++channels+examined+by+scanning+mutagenesis.">Structural similarities between glutamate receptor channels and K+ channels examined by scanning mutagenesis.</a> J Gen Phys. 117, 345-360 (2001).</li><li>Doyle, D. A., Cabral, M. o. r. a. i. s., Pfuetzner, J., Kuo, R. A., Gulbis, A., Cohen, J. M., Chait, S. L., &amp;amp, B. T., MacKinnon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+the+potassium+channel:+molecular+basis+of+K++conduction+and+selectivity.">The structure of the potassium channel: molecular basis of K+ conduction and selectivity.</a> Science. 280, 69-77 (1998).</li><li>Nishida, M., Cadene, M., Chait, B. T., MacKinnon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+a+Kir3.1-prokaryotic+Kir+channel+chimera.">Crystal structure of a Kir3.1-prokaryotic Kir channel chimera.</a> EMBO J. 26, 4005-4015 (2007).</li><li>Tao, X., Avalos, J. L., Chen, J., MacKinnon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+eukaryotic+strong+inward-rectifier+K++channel+Kir2.2+at+3.1+&amp;Aring;+resolution.">Crystal structure of the eukaryotic strong inward-rectifier K+ channel Kir2.2 at 3.1 &amp;Aring; resolution.</a> Science. 326, 1668-1674 (2009).</li></ol>

利害の衝突は宣言されません。

部位特異的突然変異誘発法は、イオンチャネルの構造と機能の関係を研究する古典的なアプローチです。それはイオンチャネルの重要な残基を変更することにより、一つのチャネルの機能に顕著な変化を観察することができるはずという前提に基づいています。対照的に、非クリティカルな位置に多くの変異は、チャネルの機能に大きな摂動なしで導入することができる。通常は、問題の残留物が最初にアラニン、トリプトファン、アミノ酸の最大の2〜突然変異が続く小さな非極性アミノ酸に変異している。残留物は、チャネルの機能に重要な場合は、両方の変異がチャネル活性を変更します。あまり本質的な残基が変異している場合、特にアラニンへの変異の影響は、しばしば平凡です。トリプトファンの変異は、トリプトファンのサイズに関連付けられている立体的な支障に起因する変化を引き起こす可能性が高くなります。多くの場合、チャンネル（アラニンやトリプトファンスキャンと呼ばれる）の断片の中アラニンやトリプトファンの構造情報が不足して、体系的な変異が重要な残基を特定するために使用することができます。別の点突然変異を持つ複数のクローンを並列比較的短い時間内に複数の残基をテストするためにできるように製造することができることに注意することは重要です。最近では、チャネルの複数の結晶構造は3,4,5利用できるようになりました。彼らは、このプロトコルで説明されているアプローチを使用してテストすることがキーとなる可能性がある残基を提案するために使用することができます。 野生型チャネル上の測定値は常にトランスフェクションし、電気生理学的実験が正しく行われたことを確実にするためのポジティブコントロールとして機能する変異体チャネルの測定を添付してください。点突然変異は、機能的な現在の不足にチャネル活性の野生型または改変された規制と同様の電流を導くことができる。測定可能な電流の欠如は、不適切な折りたたみまたは変更された人身売買に起因する可能性があります。このようなケースでは、チャネルがセルの内部留保の非機能的な細胞膜上のチャンネルとチャンネルを区別するために細胞外タグに対するビオチン化アッセイまたは免疫蛍光染色を（細胞浸透性のない）を使用して表面に正確にトラヒックのかどうかを確認すると便利な場合があります。最後に、チャネルだけでなく、チャネルの規制上の変異の影響の基底活性だけでなく、を調査することをお勧めします。チャネル機能の調節機序の異なるが、Gタンパク質、PIP2、イオン、ATP濃度、リン酸化（チロシン、セリンまたはスレオニンの変異を使用して）、ユビキチン化やSUMO化（リジンの突然変異を使用して）による規制を含む、対象のイオンチャネルの種類により研究される可能性とダイレクトチャネルオープナーまたはブロッカー。

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en"> 
 
 <div>
 <table border="1">
 <thead>
 <tr>
 <td>Material Name</td>
 <td>Type</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comment</td>
 </tr>
 </thead>
 <tbody>
 
<tr>
<td>Chemicals</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>Varies</td>
<td>&nbsp;</td>
<tr>
<td>Mini Prep Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>27104</td>
<td>&nbsp;</td>
<tr>
<td>Radiacwash</td>
<td>&nbsp;</td>
<td>Biodex</td>
<td>005-100</td>
<td>&nbsp;</td>
<tr>
<td>Poly-D-lysine hydrobromide</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>CB40210</td>
<td>Poly-L-lysine can also be used</td>
</tr>
<tr>
<td>Maxi Prep Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>12263</td>
<td>&nbsp;</td>
<tr>
<td>QuickChange XL II Site Directed Mutagenesis Kit</td>
<td>&nbsp;</td>
<td>Stratagene</td>
<td>200512-5</td>
<td>&nbsp;</td>
<tr>
<td>Lipofectamine 2000</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11668-027</td>
<td>&nbsp;</td>
<tr>
<td>Cover slips &#x02C6;&#x2026; 12 mm</td>
<td>&nbsp;</td>
<td>Warner Instruments</td>
<td>CS-12R</td>
<td>&nbsp;</td>
<tr>
<td>Capillary Glass Tubing Borosilicate-standard wall</td>
<td>&nbsp;</td>
<td>Warner Instruments</td>
<td>G150-3</td>
<td>&nbsp;</td>
<tr>
<td><a href="http://www.leica-microsystems.com/products/light-microscopes/clinical/inverted-microscopes/details/product/leica-dmi3000-b/" target="_blank">Inverted fluorescence microscope</a></td>
<td>&nbsp;</td>
<td>Leica</td>
<td>DMI3000 B</td>
<td>&nbsp;</td>
<tr>
<td>Electrophysiology</td>
<td>&nbsp;</td>
<td>Nikon/ Leica/ Zeiss</td>
<td>Any inverted DIC (Hoffmann) microscope with fluorescence</td>
<td>&nbsp;</td>
<tr>
<td>Amplifier</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>Axon Axopatch 200B amplifier</td>
<td>&nbsp;</td>
<tr>
<td>Perfusion system</td>
<td>&nbsp;</td>
<td>Warner Instruments</td>
<td>VC-6 Perfusion MM-6</td>
<td>&nbsp;</td>
<tr>
<td>Digitizer</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>Digidata 1320 digitizer</td>
<td>&nbsp;</td>
<tr>
<td>Manipulators</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>Manipulator: MP-85</td>
<td>&nbsp;</td>
<tr>
<td>Electrode Puller</td>
<td>&nbsp;</td>
<td>Narishige</td>
<td>PC10 vertical electrode puller</td>
<td>&nbsp;</td>
<tr>
<td>Isolation Table</td>
<td>&nbsp;</td>
<td>Technical Manufacturing Corporation</td>
<td>Air table</td>
<td>&nbsp;</td> </tbody>
 </table>
 </div>

mutagenesis and functional analysis of ion channels heterologously expressed in mammalian cells

我々は、イオンチャネルの点突然変異を導入することの機能的効果を研究する方法を説明します。我々は、ニューロンの興奮性を調節するための重要なGタンパク質依存性内向き整流カリウム（GIRKと呼ばれる）チャンネルを、研究しています。つの異なる哺乳類のGIRKチャネルのサブユニット（GIRK1 - GIRK4）があります - それはホモ四量体を形成するので、我々はGIRK2に焦点を当てる。このようなムスカリン性受容体（M2R）のようなGタンパク質共役受容体（GPCR）、、のさまざまな種類の刺激は、GIRKチャネルの活性化につながる。アルコールはまた、直接GIRKチャネルをアクティブにします。我々は、特にGIRKチャネルのcDNAで1つ以上のヌクレオチドを​​変更することにより、一つのアミノ酸を変異させる方法を紹介します。この変異cDNA配列は、精製された、細菌中で増幅され、点突然変異の存在はDNAシークエンシングによって確認されます。変異と野生型GIRKチャネルのcDNAは、in vitroで培養したヒト胚性腎HEK293T細胞にトランスフェクトされます。最後に、ホールセルパッチクランプ電気生理学は、異所性に発現した野生型または変異GIRKチャネルを介して巨視的なカリウム電流を研究するために使用されます。この実験では、M2R依存とアルコール依存性活性化に関するGIRK2チャンネルでL257Wの突然変異の効果を検討する。

Watch this Scientific Journal Video about Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells at JoVE.com

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

我々は、イオンチャンネルの機能上の単一の点変異の効果を研究する方法を説明します。

異種哺乳動物細胞で発現したイオンチャネルの変異導入と機能解析

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 ...

mutagenesis-functional-analysis-ion-channels-heterologously-expressed

Research

JoVE Journal

Biology

17.3K ビュー.  Salk Institute for Biological Studies. 我々は、イオンチャンネルの機能上の単一の点変異の効果を研究する方法を説明します。

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

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

アフリカツメガエルの卵母細胞で発現イオンチャネルのパッチクランプ記録

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

生物学

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

哺乳動物細胞での糖タンパク質からN -結合型糖鎖のパルス - チェイス分析

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.

哺乳動物細胞におけるDNA二重鎖切断（DSB）修復の解析

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

イオンチャネル研究のためのパッチクランプおよび灌流のテクニックで表さアフリカツメガエル卵母細胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

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).

発現とHEK - 293T細胞における組換え電位依存性イオンチャネルの電気生理学的記録用に最適化されたトランスフェクション戦略

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

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

マイクロインジェクションによる哺乳動物細胞におけるmRNA核外輸送の速度論の解析

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

内タンパク質の定方向進化のための変異導入と機能選択プロトコル E.大腸菌</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

哺乳動物細胞における細胞周期の位置の解析

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

間葉転換を誘導し、上皮の分析

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

培養細胞およびマウス組織から単離とミトコンドリアの機能解析

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...