我々は、有用な議論をカタリーナCoskerとステファニーCourchesneに感謝します。

試薬の調製<ol><li> コラーゲンコーティング ：オーブンでコラーゲンコートp35の組織培養プレートと場所37℃で分周器を給脂する前に2日間C。コラーゲンの最終濃度は0.001 N塩酸で希釈した0.71 mg / mlのになっているはず。その後、プレート当たり混合物を1 mlを加える。 </li><li> グリースローダー ：グリースローダーを埋めるために、60mlの注射器が最初にコーニングの真空グリースを封入する必要があります。グリースローダーを埋めるために注射器を使用して、45分間オートクレーブしてホイルに包んでと。 </li><li> テフロン仕切り ：分周器をそれぞれの実験後に再使用することができますが、最初の適切に洗浄する必要があります。プレートから分周器を取り外す、2日間硫酸の残りのグリースと場所のすべてをふき取ってください。酸から削除した後、水3X、20分間沸騰とリンス、、20分間ガラスP100ペトリ皿とオートクレーブ内の場所を乾燥することができます。 </li><li> N2 -メチルセルロース ：メチルセルロースの1.5グラムを秤量し、500ミリリットル瓶に入れます。攪拌棒を追加し、乾燥した上で20分間オ​​ートクレーブ（この時点から、すべての作業は無菌でなければならない）。次に、無血清培地（N 2）、500 MLSを追加し、それが溶けるまで寒い部屋でかき混ぜる。 -20℃で50mLのconicalsと凍結に分注し-20℃で作業用ストック、1mLのチューブに50 mLのconicalsの一分注して凍結するための</li><li> DRGメディア ：DMEM、5％熱不活化ウマ血清、及び1％ペニシリンストレプトマイシン。 </li><li> 100ng/mlのDRGNメディア：両方の神経成長因子（NGF）及び脳由来神経栄養因子（BDNF）の株式の濃度は1mg/mLです。 DRGのメディアへのニューロトロフィン1:10,000のそれぞれを希釈する。培養物はNGF単独で成長させることができる、これは文化の中で生き残るニューロンの補数を変更します。 注 ：必要なときは、（シトシンアラビノシド）AraCの濃度が1uMと0.3uMの最終濃度で使用されている。これは、シュワン細胞や他のグリア細胞の成長を抑制します。 </li><li> 10ng/mL DRGNメディア ：DRGのメディアで100ng/mlのDRGNメディア（1:10）希釈。 <img src="/files/ftp_upload/old/951_2008_10_27_6/951_Figure-01.png" alt="図1" width="392" height="294" /> セットアップに必要な図1。ツール </li></ol>コンパートメントチャンバーのセットアップ（解剖前に1〜2日、このプロセスを開始する） <ol><li>外側への動きとコラーゲンコーティングしたp35の皿の中央にスクラッチを行う。 </li><li>スクラッチの真ん中でN2 -メチルセルロースの30ul場所。分周器が油を塗ったされるまで、脇に料理を設定します。 </li><li>グリースのローダに23ゲージルアースタブアダプタを取り付けます。グリップテフロンの90 °の角度止血のペアで分周器とは、顕微鏡下で上向きに分周器とそれを寝かせて。グリースは、それぞれの時間は、アダプタが新しい出発点に置かれているグリースの連続的なラインが存在するようにアダプタが前のステップからグリースに挿入されていることを確認すると分周器をトレース（図を参照）。グリスが全体の分周器に適用されるとN2 -メチルセルロースは、中央の区画を超えているので、上下逆さまに準備したp35の料理のいずれかをオンにして置きます。ピンセットで皿の底を下に押してください。四隅（&quot;X&quot;によって図に示されている左上、右下、右上、左下、）で分周器の内側に押してください。 <img src="/files/ftp_upload/old/951_2008_10_9_2/951_Figure-02.png" alt="図2" width="171" height="183" /> 図2：分周器を給脂する手順 注 ：これは、グリースが料理と完全なシールを作るが、過剰な圧力が付加されている場合、軸索は側の区画にまたがることがないようにしっかりと十分に押すことが重要です。 、止血を拾うそれを裏返して分周器をアンクランプ。最後に、しっかりと中間のコンパートメントの底部に焦点を当てて顕微鏡下に取り付けられた分圧器を使って皿を置く。グリースのローダーで、細胞が中央コンパートメント内に配置されると、彼らが漏れないことができるように小さな障壁（0.25 cm）を行う。 </li><li>複数の文化、サイド区画のそれぞれの場所のDRGのメディアと細胞が維持されるのインキュベーター内で場所を設定した後。文化は数時間放置し、漏れをチェックすることができます。メディアが真ん中のコンパートメントに流出している場合には、文化は使用不能です。 注 ：いくつかが漏れになるので最初にこのテクニックを学ぶとき、それは、実験に必要な数よりも多くの文化を設定することが重要です。 <img src="/files/ftp_upload/old/951_2008_10_9_3/951_Figure-03.png" alt="図3" width="254" height="191" /> 図3：&quot;グッドVS漏れ&quot;文化 </li></ol>コンパートメントの文化の維持DRGニューロン<ol><li> 1日目 ：100ng/mlのDRGNメディア+ AraCのある側のコンパートメントにDRGメディアを交換してください。解剖を行い、中央の区画（100,000セル）にセルを追加。 </li><li> 2日目 ：メディアセンターのコンパートメントを持つグリースの障壁と交換流体を介して流れるまで、テフロンの分周器の外側に10ng/mLメディア+ AraCのを追加。 </li><li> 3日目 ：10ng/mL DRGNはAraCのを省略してAraCのとサラウンドを省略100ng/mlのDRGNする側のコンパートメントでメディアを交換してください。 </li><li> 6日目 ：1ng/mL + AraCのとDRGメディア+ AraCのあるサラウンドにサイドコンパートメント内のメディアを交換してください。 </li><li> 9日目 ：実験のために使用して下さい。 <img src="/files/ftp_upload/old/951_2008_10_9_5/951_Figure-04.png" alt="図4" width="267" height="148" /> 図4：細胞体と軸索遠位のIHC画像 注：メディアを変更する場合、それは各サイド区画の上部から液体を吸引することが重要です。また、唯一のサラウンドから、中間のコンパートメント自体からメディアを変更していない、そして中央にグリスの障壁を越えて、流れてみましょうね。 </li></ol>

<ol>
	<li>Campenot, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Independent+Control+of+the+Local+Environment+of+Somas+and+Neurites.">Independent Control of the Local Environment of Somas and Neurites.</a> Methods in Enzymology. 58, 302-307 (1979).</li><li>Watson, F. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophins+use+the+Erk5+pathway+to+mediate+a+retrograde+survival+response.">Neurotrophins use the Erk5 pathway to mediate a retrograde survival response.</a> Nature Neuroscience. 4, 981-988 (2001).</li><li>Heerssen, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynein+motors+transport+activated+Trks+to+promote+survival+of+target-dependent+neurons.">Dynein motors transport activated Trks to promote survival of target-dependent neurons.</a> Nature Neuroscience. 7, 596-603 (2004).</li><li>Taylor, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+culture+platform+for+CNS+axonal+injury,+regeneration+and+transport.">A microfluidic culture platform for CNS axonal injury, regeneration and transport.</a> Nature Methods. 2, 599-605 (2006).</li><li>Park, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+culture+platform+for+neuroscience+research.">Microfluidic culture platform for neuroscience research.</a> Nat Protoc. 4, 2128-2136 (2006).</li><li>Ure, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrograde+transport+and+steady-state+distribution+of+125I-nerve+growth+factor+in+rat+sympathetic+neurons+in+compartmented+cultures.">Retrograde transport and steady-state distribution of 125I-nerve growth factor in rat sympathetic neurons in compartmented cultures.</a> J Neuroscience. 4, 1282-1290 (1997).</li></ol>

このビデオでは、我々は培養DRGニューロンにおける使用のためにコンパートメント室を準備し、維持する方法を示している。正しく行えば、このシステムは、ニューロトロフィンは、長い軸索を介して通知されるメカニズムを研究するために、軸索から細胞体を分離することができます。コンパートメントの間に流体分離があるので、それは他のコンパートメントが影響を受けることなく、一区画の選択的?...

<!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>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>collagen</td>
<td>Reagent</td>
<td>BD Biosciences</td>
<td>354249</td>
<td>&nbsp;</td>
<tr>
<td>N2-methylcellulose 400CPS</td>
<td>Reagent</td>
<td>Sel-Win Chemicals</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Teflon divider</td>
<td>Other</td>
<td>Tyler Research</td>
<td>CAMP10</td>
<td>many other types of dividers are available</td>
</tr>
<tr>
<td>Pin rake</td>
<td>Tool</td>
<td>Tyler Research</td>
<td>Camp-PR</td>
<td>&nbsp;</td>
<tr>
<td>Grease loader</td>
<td>Tool</td>
<td>Tyler Research</td>
<td>Camp-GLSS</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>Reagent</td>
<td>Fisher</td>
<td>MT10017CV</td>
<td>&nbsp;</td>
<tr>
<td>NGF</td>
<td>Reagent</td>
<td>Peprotech</td>
<td>450-01</td>
<td>&nbsp;</td>
<tr>
<td>BDNF</td>
<td>Reagent</td>
<td>Peprotech</td>
<td>450-02</td>
<td>&nbsp;</td>
<tr>
<td>High vacuum grease</td>
<td>Reagent</td>
<td>Fisher</td>
<td>14-635-5D</td>
<td>&nbsp;</td>
<tr>
<td>AraC</td>
<td>Reagent</td>
<td>Sigma</td>
<td>C-1768</td>
<td>&nbsp;</td>
<tr>
<td>23 gauge luer stub adapter</td>
<td>Tool</td>
<td>Fisher</td>
<td>427565</td>
<td>&nbsp;</td>
<tr>
<td>90&deg; angle hemostats</td>
<td>Tool</td>
<td>Roboz</td>
<td>RS-7035</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

preparation and maintenance of dorsal root ganglia neurons in compartmented cultures

ニューロンは、遠くのターゲット由来の成長因子がニューロンの生存と機能に必要な標的組織を、支配するために細胞体から削除されている軸索のプロセスを拡張します。ニューロトロフィンは、特に感覚神経の神経支配の生存と分化を維持することが義務付けられていますが、これらの標的由来神経栄養因子は神経支配ニューロンの細胞体への通信方法の質問は、30年以上にわたり活発な研究分野となっている。ニューロトロフィンのシグナルが細胞体に到達する方法の最も一般的に受け入れられているモデルは、シグナルが軸索に沿って逆行性にこの信号を運ぶエンドソームことを提案している。逆行性輸送を研究するために、培養系は、もともと細胞体がそれらの軸索から隔離されているロバートCampenot、によって考案されました。培養感覚ニューロンの反復するターゲット由来神経栄養因子のin vivoで、次のリリースで発生するニューロンの端末の選択刺激のためにこれらのコンパートメントチャンバーの準備のテクニック。長距離微小管依存性の逆行輸送を必要とする逆行性シグナル伝達事象は、神経変性疾患の治療のために重要な意味を持っている。

Watch this Scientific Journal Video about Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures at JoVE.com

Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

ここでは、後根神経節の培養感覚ニューロンについてはコンパートメント室を準備し、維持するテクニックについて説明します。

コンパートメント文化の後根神経節ニューロンの準備とメンテナンス

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for ...

preparation-maintenance-dorsal-root-ganglia-neurons-compartmented

Research

JoVE Journal

Biology

22.3K ビュー.  Dana Farber Cancer Institute. ここでは、後根神経節の培養感覚ニューロンについてはコンパートメント室を準備し、維持するテクニックについて説明します。

ビデオ: Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

Midgastrulaステージショウジョウバエの胚からの神経培養の準備

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

生物学

Preparation of Dissociated Mouse Cortical Neuron Cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.

解離マウス皮質ニューロンの培養の準備

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed protocol for biolistic transfection of rat hippocampal slices, from the initial preparation of DNA coated bullets to the final shooting of the organotypic slice cultures using a gene gun. Gene gun transfection is an efficient and easy means of transfecting neurons and is especially useful for fluorescently labeling a small subset of cells in tissue slice. In this video, we first outline the steps required to coat gold particles with DNA. We next demonstrate how to line the inside of plastic tubing with the gold/DNA bullets, and how to cut this tubing to obtain the plastic cartridges for loading into the gene gun. Finally, we perform biolistic transfection of rat hippocampal slice cultures, demonstrating handling of the Bio-Rad Helios gene gun, and offering trouble shooting advice to obtain healthy and optimally transfected tissue slices.  

We describe a method for preparing DNA coated gold bullets and demonstrate the use of such bullets to biolistically transfect neurons in cultured hippocampal slices.

遺伝子銃の箇条書きとスライス培養における神経細胞の微粒子銃トランスフェクションの準備

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed ...

Preparation of 2-dGuo-Treated Thymus Organ Cultures

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus.

2 - dGuo処理した胸腺器官培養の準備

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play ...

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG)

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide sympathetic innervations for the head and neck regions and they constitute a well-characterized and relatively homogeneous population (4).  Sympathetic neurons are dependent on nerve growth factor (NGF) for survival, differentiation and axonal growth and the wide-spread availability of NGF facilitates their culture and experimental manipulation (2, 3, 6).  For these reasons, cultured sympathetic neurons have been used in a wide variety of studies including neuronal development and differentiation, mechanisms of programmed and pathological cell death, and signal transduction (1, 2, 5, and 6).  Dissecting out the SCG from newborn rats and culturing sympathetic neurons is not very complicated and can be mastered fairly quickly.  In this article, we will describe in detail how to dissect out the SCG from newborn rat pups and to use them to establish cultures of sympathetic neurons.  The article will also describe the preparatory steps and the various reagents and equipment that are needed to achieve this.

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

This is a protocol describing how to isolate and culture primary sympathetic neurons from superior cervical ganglia (SCG) of newborn rat pups.

ラット上頚神経節（SCG）から培養交感神経のためのプロトコル

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide ...

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.
An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.
In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

の準備アメフラシ感覚運動神経細胞の培養

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

ヒト多能性のための代替培養はセル生産、メンテナンス、および遺伝子解析幹

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Lateral Root Inducible System in Arabidopsis and Maize

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is however tedious, because it occurs only in a few cells inside the root and in an unpredictable manner. To circumvent this problem, a Lateral Root Inducible System (LRIS) has been developed. By treating seedlings consecutively with an auxin transport inhibitor and a synthetic auxin, highly controlled lateral root initiation occurs synchronously in the primary root, allowing abundant sampling of a desired developmental stage. The LRIS has first been developed for Arabidopsis thaliana, but can be applied to other plants as well. Accordingly, it has been adapted for use in maize (Zea mays). A detailed overview of the different steps of the LRIS in both plants is given. The combination of this system with comparative transcriptomics made it possible to identify functional homologs of Arabidopsis lateral root initiation genes in other species as illustrated here for the CYCLIN B1;1 (CYCB1;1) cell cycle gene in maize. Finally, the principles that need to be taken into account when an LRIS is developed for other plant species are discussed.

Lateral Root Inducible System in Arabidopsis and Maize

The Lateral Root Inducible System (LRIS) allows for synchronous induction of lateral roots and is presented for Arabidopsis thaliana and maize.

側根誘導系で<em&gt;シロイヌナズナ</em&gt;とトウモロコシ

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is ...

Divergence of Root Microbiota in Different Habitats based on Weighted Correlation Networks

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, which can effectively explore the interaction relationship or co-occurrence model of different microbial species in different environments. The purpose of this manuscript is to provide details on how to use the weighted correlation network algorithm to analyze different co-occurrence networks that may occur in microbial communities due to different ecological environments. All analysis of the experiment is performed in the WGCNA package. WGCNA is an R package for weighted correlation network analysis. The experimental data used to demonstrate these methods were microbial community data from the NCBI (National Center for Biotechnology Information) database for three niches of the rice (Oryza sativa) root system. We used the weighted correlation network algorithm to construct co-abundance networks of microbial community in each of the three niches. Then, differential co-abundance networks among endosphere, rhizoplane and rhizosphere soil were identified. In addition, the core genera in network were obtained by the "WGCNA" package, which plays an important regulated role in network functions. These methods enable researchers to analyze the response of microbial network to environmental disturbance and verify different microbial ecological response theories. The results of these methods show that the significant differential microbial networks identified in the endosphere, rhizoplane and rhizosphere soil of rice.

Network analysis was applied to evaluate the association of various ecological microbial communities, such as soil, water and rhizosphere. Presented here is a protocol on how to use the WGCNA algorithm to analyze different co-occurrence networks that may occur in the microbial communities due to different ecological environments.

重み付け相関ネットワークに基づく異なる生息地における根内微生物叢の発散

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

根の滲出液中の根茎細菌の化学誘引物質の迅速な同定のための改良された気化症アッセイ

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...