我々はこの作業の支援のためCIRMに感謝します。細胞株は、寛大にマウントサイナイ病院、トロント、オンタリオ州で博士アンドラーシュNagy氏から寄贈されました。

1。 Shrinky -ディンク金型を作る<ol><li>良い定義のプリンターを使用してshrinky -ディンクのシート上に所望のパターンを印刷します。 </li><li> 163で焼くshrinky -ディンクシート℃で約10分間、または完全に縮小し、定期的な形状を取得した時まで。 </li><li> shrinky -ディンクの金型冷却された後、イソプロパノール浴に浸し、それは完全な表面はほとんどカバーされるまで。 </li><li>慎重に、金型の上にいくつかのアセトンをスプレーし、容器を数回振る。アセトンの過剰を洗い流すとshrinky -カビがきれいになってまで、この手順を数回繰り返すよりイソプロパノール追加。 </li><li>残りの有機溶剤を洗い落とすために10分間蒸留水に浸けてカビ。 </li><li>エアクリーンshrinkyモールド。この意志コンパクトなインク163℃で約5分間、それを再加熱し、残りの溶剤を蒸発させる。 </li></ol> 2。 PDMSマイクロウェルの作成<ol><li> 10時01 PDMS /硬化剤の混合物を準備し、数分間激しく振とうする。 </li><li>小さなペトリ皿で​​行わshrinky -ディンクのモールド。それは金型表面上の約1 / 2 cmに達するまでPDMSの混合物を注ぐ。 </li><li> PDMSの混合物からすべての気泡を除去するため真空鐘の下に皿を置きます。 </li><li> 70℃のオーブンで行わ皿° C、一晩。 </li><li>金型から固体PDMSを遮断し、単に圧力を適用することにより、ガラススライドにバインドします。 </li><li>それは、PDMSとの間でincrustedインクの残基を有しているため、最初にマイクロチップを捨てる。 </li><li>インクフリーであると、より定義された形状を持つ別のチップを製造するには、この手順を繰り返します。 </li><li>クリーンマイクロチップの70％エタノール溶液を用いた。それを殺菌するために10分間UV光源の下に置きます。 </li></ol> 3。マイクロウェルにトラップ細胞<ol><li>細胞をカウントし、所望の濃度（希望するどのように多くの井戸の最初のセルに応じて）に培地でそれらを希釈する。例えば、ウェルあたり約10〜15セル（平均= 11、SD = 5.4、積載率= 93％）を得るために、我々は、8 × 10 4細胞/ mlの濃度を使用していました。 17 × 10 4細胞/ mlの濃度の場合、我々は確実によくあたり25〜35セル（平均= 27.17857 SDは= 7.7、積載率= 100％）得ることができる。 </li><li>慎重に固化したPDMSの塩基を含む50 ml遠心チューブにマイクロチップを置きます。 </li><li>細胞溶液の2JDP ml程度追加。 </li><li> 5 760 rpmで分、4℃のための遠心分離機</li><li>過剰の溶液からピペットで慎重にPBS 1Xソリューションをマイクロウェルを洗う。 </li><li>遠心管からチップを取り出しながら、注意しながら、小さなペトリ皿の中でのマイクロウェルを置きます。 </li><li> 1 X PBS溶液を用いて細胞の過剰を洗ってください。 </li><li>ウェルあたりの細胞の目的の数を確認するために倒立顕微鏡下でマイクロウェルチップを置きます。 </li><li>標準的な条件下でマイクロウェルを含む細胞をインキュベートする。 <img src="/files/ftp_upload/old/692_2008_7_21_1/692_Figure-01.png" alt="図- 01" width="350" height="183" /></li></ol> 4。細胞のインキュベーション<ol><li>ラボでの通常のEBのプロトコルに従ってください。 </li><li>徐々にチャンバーの側からメディアを変更してください。マイクロウェルに細胞を乱すことを避ける。 </li></ol>

<ol>
	<li>Keller, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+differentiation+of+embryonic+stem+cells.">In vitro differentiation of embryonic stem cells.</a> Curr Opin Cell Biol. 7, 862-869 (1995).</li><li>Doetschman, T. C., Eistetter, H., Katz, M., Schmidt, W., Kemler, 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+in+vitro+development+of+blastocyst-derived+embryonic+stem+cell+lines:+Formation+of+visceral+yolk+sac,+blood+islands+and+myocardium.">The in vitro development of blastocyst-derived embryonic stem cell lines: Formation of visceral yolk sac, blood islands and myocardium.</a> Journal of Embryology and Experimental Morphology. 87, 27-45 (1985).</li><li>Park, J., Cho, C. H., Parashurama, N., Li, Y., Berthiaume, F., Toner, M., Tilles, A. W., Yarmush, 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=Microfabrication-based+modulation+of+embryonic+stem+cell+differentiation.">Microfabrication-based modulation of embryonic stem cell differentiation.</a> Lab Chip. 7, 1018-1028 (2007).</li><li>Koike, M., Sakaki, S., Amano, Y., Kurosawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+embryoid+bodies+of+mouse+embryonic+stem+cells+formed+under+various+culture+conditions+and+estimation+of+differentiation+status+of+such+bodies.">Characterization of embryoid bodies of mouse embryonic stem cells formed under various culture conditions and estimation of differentiation status of such bodies.</a> J Biosci Bioeng. 104, 294-299 (2007).</li><li>Hwang, N. S., Varghese, S., Elisseeff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+differentiation+of+stem+cells.">Controlled differentiation of stem cells.</a> Advanced Drug Delivery Reviews. 60, 199-204 (2008).</li><li>Adelman, C. A., Chattopadhyay, S., Bieker, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+BMP/BMPR/Smad+pathway+directs+expression+of+the+erythroid-specific+EKLF+and+GATA1+transcription+factors+during+embryoid+body+differentiation+in+serum-free+media.">The BMP/BMPR/Smad pathway directs expression of the erythroid-specific EKLF and GATA1 transcription factors during embryoid body differentiation in serum-free media.</a> Development. 129, 539-549 (2002).</li><li>Tanaka, N., Takeuchi, T., Neri, O. V., Sills, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-assisted+blastocyst+dissection+and+subsequent+cultivation+of+embryonic+stem+cells+in+a+serum/cell+free+culture+system:+applications+and+preliminary+results+in+a+murine+model.">Laser-assisted blastocyst dissection and subsequent cultivation of embryonic stem cells in a serum/cell free culture system: applications and preliminary results in a murine model.</a> J Transl Med. 4, (2006).</li></ol>

Weは、微細加工井戸（Shrinky - DINKSから成形された）へのセルの定義済みの数値をロードし、胚様体の開発のためにそれらを維持するために、シンプル、迅速、かつスケーラブルな培養法を開発した。最後に、これらの細胞は、さらに分析と実験のために簡単にアクセス可能です。このメソッドは、どの研究室に適していると我々はフォトリソグラフィの必要性を未然に防ぐため、専用機器を必要としません?...

KO DMEM：40.8ミリリットル、81.6ミリリットルの15％KSR：7.5ミリリットル、15ミリリットル× 1ペン、連鎖球菌：0.5ミリリットル、1ミリリットル2mMのL -グルタミン：;：（100ミリリットル50ミリリットル調製した全メディアのための：）MESCメディア（マクロスキーラボプロトコル）のための0.5ミリリットル、1ミリリットルNEAA：0.5ミリリットル、1ミリリットルLIF：100ul、200ul BMP - 4（10ng/ml）：50UL、100ulの希薄化後BME：。50UL、100ul（PBSとシリンジフィルター5mlのために滅菌濾過BMEの35ulを追加する滅菌破棄2週間後。溶液中の最終濃度は0.1 mMのです）

shrinky-dink hanging drops: a simple way to form and culture embryoid bodies

胚様体（EB）は、胚性幹細胞の集合体です。これらの集計を作成する最も一般的な方法は、ハンギングドロップ法、ウェルプレートに細胞を任意の数のピペッティングの面倒なアプローチです。互いの接近に強制的に幹細胞間の相互作用は、EBSの生成を促進。各ウェル内のメディアを手動で毎日交換する必要があるため、このアプローチは、手作業の多いです。 同一の条件下で培養するとさらに、細胞 - 細胞、細胞可溶性因子の相互作用は、pH、及び酸素の可用性を含む、環境パラメータは、EBの大きさの関数である可能性があるため、従来の吊り下げ降下から得られた細胞集団は、さらに劇的に異なることがあります。最近の研究では、実際に集計を形成する細胞の初期数は、幹細胞の分化に大きな影響を持つことができることが示されている。 Weは、微細加工のウェルに細胞の定義済みの数値をロードし、胚様体の開発のためのそれらを維持するために、シンプル、迅速、かつスケーラブルな培養法を開発した。最後に、これらの細胞は、さらに分析と実験のために簡単にアクセス可能です。このメソッドは、どの研究室に適していると、専用機器を必要としません。私たちは、赤色蛍光マウスの細胞株（129S6B6 - F1）を使用して胚様体を作成して、このメソッドを示しています。

Watch this Scientific Journal Video about Shrinky-Dink Hanging Drops: A Simple Way to Form and Culture Embryoid Bodies at JoVE.com

Shrinky-Dink Hanging Drops: A Simple Way to Form and Culture Embryoid Bodies

我々は微細加工のウェルに細胞の事前定義された数値をロードし、胚様体の開発のためにそれらを維持するために簡単かつ迅速な方法を示しています。

滴をハンギングShrinky -ディンク：胚の体を形成し、文化に簡単

Embryoid bodies (EB) are aggregates of embryonic stem cells. The most common way of creating these aggregates is the hanging drop method, a laborious ...

shrinky-dink-hanging-drops-simple-way-to-form-culture-embryoid

Research

JoVE Journal

Biology

14.9K ビュー.  University of California Merced - UC Merced. 我々は微細加工のウェルに細胞の事前定義された数値をロードし、胚様体の開発のためにそれらを維持するために簡単かつ迅速な方法を示しています。

ビデオ: Shrinky-Dink Hanging Drops: A Simple Way to Form and Culture Embryoid Bodies

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

パイロシーケンシング：正確なジェノタイピングのための簡単​​な方法

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

生物学

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

初期のニワトリ胚をex vivo（新文化）の文化のための方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Analysis of Pluripotent Stem Cells by using Cryosections of Embryoid Bodies

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage early mammalian embryos 1. A crucial stage in the differentiation of ES cells is the formation of embryoid bodies (EBs) aggregates 2, 3. EB formation is based on spontaneous aggregation when ES cells are cultured in non adherent plates. Three-dimensional EB recapitulates many aspects of early mammalian embryogenesis and differentiate into the three germ layers: ectoderm, mesoderm and endoderm 4.
Immunofluorescence and in situ hybridization are widely used techniques for the detection of target proteins and mRNA present in cells of a tissue section 5, 6, 7. Here we present a simple technique to generate high quality cryosections of embryoid bodies. This approach relies on the spatial orientation of EB embedding in OCT followed by the cryosection technique. The resulting sections can be subjected to a wide variety of analytical procedures in order to characterize populations of cells containing certain proteins, RNA or DNA. In this sense, the preparation of EB cryosections (10&mu;m) are essential tools for histology staining analysis (e.g. Hematoxilin and Eosin, DAPI), immunofluorescence (e.g. Oct4, nestin) or in situ hybridization. This technique can also help to understand aspects of embryogenesis with regards to the maintenance of the tri-dimensional spherical structure of EBs.

Pluripotent stem cells growing in suspension differentiate into embryoid bodies (EBs). Here we demonstrate how to obtain high quality EB cryosections useful for studying cellular and molecular aspects of embryogenesis, while preserving their organization as aggregates.

胚様体の凍結切片を使用して、多能性幹細胞の解析

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage early mammalian embryos 1. A crucial stage in the ...

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses1.
 Various strategies such as Staput2,3, centrifugal elutriation1, and flow cytometry (FC)4,5 have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies. 
It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates - which tend to form because of the syncytial nature of the seminiferous epithelium6,7 - and lack cell clumps1 . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones1 . On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types1,8 . 
	The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting4, and ulterior gene expression studies9 .

A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.

精巣細胞懸濁液の調製のためのシンプルで効率的な手法

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

蛍光DNAための簡単​​な方法<em&gt;その場で</em&gt;ぺしゃんこ染色体へのハイブリダイゼーション

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly ...

Using Mouse Mammary Tumor Cells to Teach Core Biology Concepts: A Simple Lab Module

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in preparation for biomedical, graduate and professional programs or careers in science. To address this, a simple laboratory module was devised to teach the concepts of cell division, cellular communication and cancer through the application of animal cell culture techniques. Here the mouse mammary tumor (MMT) cell line is used to model for breast cancer. Students learn to grow and characterize these animal cells in culture and test the effects of traditional and non-traditional chemotherapy agents on cell proliferation. Specifically, students determine the optimal cell concentration for plating and growing cells, learn how to prepare and dilute drug solutions, identify the best dosage and treatment time course of the antiproliferative agents, and ascertain the rate of cell death in response to various treatments. The module employs both a standard cell counting technique using a hemocytometer and a novel cell counting method using microscopy software. The experimental procedure lends to open-ended inquiry as students can modify critical steps of the protocol, including testing homeopathic agents and over-the-counter drugs. In short, this lab module requires students to use the scientific process to apply their knowledge of the cell cycle, cellular signaling pathways, cancer and modes of treatment, all while developing an array of laboratory skills including cell culture and analysis of experimental data not routinely taught in the undergraduate classroom.

A feasible laboratory module for biology undergraduates that explores advanced cellular and molecular concepts using animal cell culture is described. Students grow, characterize and manipulate a breast cancer cell model by exposure to chemotherapy agents. Cell viability is assayed through cell counting using both a standard and novel method.

コア生物学の概念を教えるために、マウス乳腺腫瘍細胞を用いた：簡単なラボ·モジュール

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

核酸の濾過単離：簡易迅速DNA抽出方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain (RTA) Trafficking in Yeast

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ultimately kill. Such A/B toxins generally consist of an enzymatically active Asubunit (e.g., ricin toxin A (RTA)) and one or more cell binding Bsubunit(s), which are responsible for toxin binding to specific cell surface receptors. Our current knowledge of how A/B toxins are capable of efficiently intoxicating cells helped scientists to understand fundamental cellular mechanisms, like endocytosis and intracellular protein sorting in higher eukaryotic cells. From a medical point of view, it is likewise important to identify the major toxin trafficking routes to find adequate treatment solutions for patients or to eventually develop therapeutic toxin-based applications for cancer therapy.
Since genome-wide analyses of A/B toxin trafficking in mammalian cells is complex, time-consuming, and expensive, several studies on A/B toxin transport have been performed in the yeast model organism Saccharomyces cerevisiae. Despite being less complex, fundamental cellular processes in yeast and higher eukaryotic cells are similar and very often results obtained in yeast can be transferred to the mammalian situation.
Here, we describe a fast and easy to use reporter assay to analyze the intracellular trafficking of RTA in yeast. An essential advantage of the new assay is the opportunity to investigate not only RTA retro-translocation from the endoplasmic reticulum (ER) into the cytosol, but rather endocytosis and retrograde toxin transport from the plasma membrane into the ER. The assay makes use of a reporter plasmid that allows indirect measurement of RTA toxicity through fluorescence emission of the green fluorescent protein (GFP) after in vivo translation. Since RTA efficiently prevents the initiation of protein biosynthesis by 28S rRNA depurination, this assay allows the identification of host cell proteins involved in intracellular RTA transport through the detection of changes in fluorescence emission.

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain RTA Trafficking in Yeast

In the manuscript, we describe the use of a yeast-based fluorescence reporter assay to identify cellular components involved in the trafficking and killing processes of the cytotoxic A subunit of the plant toxin ricin (RTA).

リシン毒素チェーン (RTA) 酵母の人身売買に必要な細胞成分を識別するために簡単な蛍光レポーターの試金

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ...

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. Therefore, it is important to establish an efficient transient gene expression system in soybean. Here, we report a simple protocol for the preparation of soybean protoplasts and its application for transient functional analyses. We found that young unifoliate leaves from soybean seedlings resulted in large quantities of high quality protoplasts. By optimizing a PEG-calcium-mediated transformation method, we achieved high transformation efficiency using soybean unifoliate protoplasts. This system provides an efficient and versatile model for examination of complex regulatory and signaling mechanisms in live soybean cells and may help to better understand diverse cellular, developmental and physiological processes of legumes.

We developed a simple and efficient protocol for the preparation of large quantities of soybean protoplasts to study complex regulatory and signaling mechanisms in live cells.

簡便な分離大豆プロトプ ラストと一過的遺伝子発現解析への応用

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. ...

A Proximal Culture Method to Study Paracrine Signaling Between Cells

Intercellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and development. Paracrine or juxtacrine signaling mediates such interactions. The use of a conditioned medium and coculture studies are the most common methods to discriminate between these two types of interactions. However, the effect of localized high concentrations of secreted factors in the microenvironment during the paracrine interactions is not accurately recapitulated by conditioned medium and, thus, may lead to imprecise conclusions. To overcome this problem, we have devised a proximal culture method to study paracrine signaling. The two cell types are grown on either surface of a 10 &#181;m-thick polycarbonate membrane with 0.4 &#181;m pores. The pores allow the exchange of secreted factors and, at the same time, inhibit juxtacrine signaling. The cells can be collected and lysed at the endpoint to determine the effects of the paracrine signaling. In addition to allowing for localized concentration gradients of secreted factors, this method is amenable to experiments involving prolonged periods of culture, as well as the use of inhibitors. While we use this method to study the interactions between ovarian cancer cells and the mesothelial cells they encounter at the site of metastasis, it can be adapted to any two adherent cell types for researchers to study paracrine signaling in various fields, including tumor microenvironment, immunology, and development.

Paracrine and juxtacrine cellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and development. Here, a proximal culture method is used to study paracrine signaling where the localized concentrations of the secreted factors are maintained while preventing direct cellular contact.