養子DTHの誘導 Joveの資料を参照してください ： <a href="/index/Details.stp?ID=325">ラットにおける養子遅延型過敏症の誘導とモニタリング</a> クリスティンビートン、ジョージK. Chandy 生理学と生物物理学専攻、カリフォルニア大学アーバイン校 J.可視化実験、第8号ここで、我々は、貪食抗原提示細胞の可視化を可能にするために非標識抗原と1:1の比率で抗原（卵白アルブミン）テキサスレッドコンジュゲートさを使用して、このプロトコルを変更しました。 耳組織の収穫<ol><li>あなたの承認された動物のプロトコルに記載されている手順で、目的の時間の時点でラットを安楽死させる。ここでは、イソフルラン麻酔薬の過剰摂取を、使用してください。これは、よく換気フード内にある密閉容器内で行われます。動物は、胸腔を開いて心臓を切断することによってのいずれかの斬首によって死んでいることを確認します。 </li><li>大きなハサミを使用すると、可能な限り動物の体に近づけるシングルカット、（図1）で耳を取り外します。 <img src="/files/ftp_upload/old/907_2008_8_13_0/Figure_1.jpg" alt="Figure_1.jpg" /> 図1。ラットの耳の除去 </li><li>氷冷した1 × PBSまたはRPMI - 1640の約10 mlを入れた15 mlコニカルチューブに耳を置きます。あなたがイメージングのために組織を準備する準備が整うまで、氷上に組織サンプルを保管してください。 </li><li>できるだけ早く画像組織、我々は、収穫し、すぐに画像化組織と比較した場合、氷上で1〜2時間は、組織の細胞の運動性には目に見える効果がないこと、しかし、見つける。 </li></ol>イメージングのための組織の準備表皮と真皮層のI.除去 *注意 ：深い真皮層はそのまま保持されている必要があります。正しく行えば、大きな血管が損なわれることと耳の軟骨は公開されません。は炎症（コントロール条件）がない場合は特にこれは、難しいテクニックであり、それは解剖顕微鏡を使用すると便利です。 <ol><li> 10cmの組織培養皿の円錐管から組織とメディアの両方を置きます。 </li><li>手袋をはめた手では（図2）を上に向けて耳の背側と耳（遠位）の先端に軽くラットの耳をホールドして。 <img src="/files/ftp_upload/old/907_2008_8_13_1/Figure_2.jpg" alt="Figure_2.jpg" /> 図2。耳オフトリム毛皮 </li><li>深い真皮から真皮を分離するために、閉じたデュモン＃5はさみの先端を使用しながら、耳の上、安定した組織で、親指で。また、耳の端が皮膚/深い真皮のオーバーハングを持つ場合、これはピンセットで把握することができ、ゆっくりと組織の下にある（図3）からこれを分離するために引っ張った。 <img src="/files/ftp_upload/old/907_2008_8_13_2/Figure_3.jpg" alt="Figure_3.jpg" /> 図3。真皮上層の除去 </li><li>一度、皮膚の小さな領域が交互に優しく除去し、外れた皮膚を剥がしに真皮層と鉗子の間でカットするはさみを使って分離の手順を繰り返します、下にある組織から分離されています。 </li><li> 5 × 5 mmまたは抗原の注射の最初の部位（図4）から少なくとも3 mmである耳組織の大きな面積を公開しています。 <img src="/files/ftp_upload/old/907_2008_8_13_3/Figure_4.jpg" alt="Figure_4.jpg" /> 図4。イメージングに曝露された組織 </li></ol> II。イメージングのステージに組織を保護<ol><li>準備組織よりも少し大きいサイズのプラスチック製カバースリップをカット。 </li><li> 3M Vetbondスライド全体™組織セーフ接着剤の薄層を広げる。 </li><li>レンズの紙やキムの組織の下側、準備組織の隅をつかみ（あなたがイメージする可能性がない場合）、メディアから削除し、DABは、余分なメディアを削除するに拭いてください。 </li><li>接着剤カバーしたスライド（すぐにウェットティッシュでVetbond治療法）に組織の端をタッチ。ゆっくりと（図5）スライドに触れないように最後は反対側で、フラットな組織を伸ばす <img src="/files/ftp_upload/old/907_2008_8_13_4/Figure_5.jpg" alt="Figure_5.jpg" /> 図5。スリップをカバーするために組織をマウントします。 </li><li>反転組織サンプルでVetbond™を治す/スライドと組織とスライドは、45度の角度でダウンとほぼ直面しているように、メディアの貯水池に浸漬。角度を上下逆さまに組織を保持すると、曝露された組織の表面に硬化から余分な接着剤を防ぐことができます。組織の表面上に接着剤は、レーザー（図6）をブロックします。 <img src="/files/ftp_upload/old/907_2008_8_13_5/Figure_6.jpg" alt="Figure_6.jpg" /> 図6。 vetbond組織GLUを治すメディアに浸漬することによって、電子 注 ：ステップ2-5は、マスターに少し練習がかかる場合があります。初めて試みる前に破棄または余分な組織片で練習してみてください。 </li><li> DABスライドの底にシリコングリスを少量。 </li><li>灌流チャンバ内のスライドを置きます。灌流のメディアは、37 ° Cを流れると組織を追加する前に酸素をされていることを確認します。ゆっくりと灌流チャンバーの底部とプラスチック製のカバーガラスの間にシリコングリスでシールを作るスライドの端を押し下げます。 </li></ol> III。二光子イメージング<ol><li>明視野透過光を使用して、組織の上に焦点を当てる。ライトのすべての電源を切ります。 </li><li> 、レーザーのようにして多光子顕微鏡のシステムで必要とされる光電子増倍管を入れます。 </li><li>高秩序の繊維状タンパク質で第二高調波発生（SHG）（コラーゲンおよびミオシン）は、励起レーザーの半波長の信号を生成する。我々は、顕微鏡の&quot;青&quot;のチャンネルを使って視覚化できる450 nmでSHGにつながる、900nmの励起を使用してください。世代のSHGと非線形（二光子）のプロセスであるので、蛍光の二光子励起の場合と同様の光学&quot;切片&quot;の効果を提供します。並列で実行される線維が可視化されることはありませんが、SHGのピーク効率は、線維がレーザーに対して垂直になるときに発生します1。 </li><li>細胞をたっぷり使って良好な結像領域を見つけ、細胞の大部分が中央にあり、イメージングを開始するようにして取得面積を設定します。注：DTH反応では、T細胞および他の細胞浸潤はしばしば他の領域は、セル2の浸潤性を欠いている間に一緒に集中して発見された。そのため、画像への最適な領域を見つけるために組織を検索し、時間がかかることがあります。 </li><li>撮像中にセルの速度と極性を評価することにより細胞の生存を確認してください。最小限の写真 - ダメージを確保するために許容可能な画質を提供する最小のレーザパワーを使用してください。 </li><li>必要に応じて新しい組織の準備のために交換する。 </li></ol>

<ol>
	<li>Koo, G. C., 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=Blockade+of+the+voltage-gated+potassium+channel+Kv1.3+inhibits+immune+responses+in+vivo.">Blockade of the voltage-gated potassium channel Kv1.3 inhibits immune responses in vivo.</a> J Immunol. 158, 5120-5128 (1997).</li><li>Gaga, M., Frew, A. J., Varney, V. A., Kay, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eosinophil+activation+and+T+lymphocyte+infiltration+in+allergen-induced+late+phase+skin+reactions+and+classical+delayed-type+hypersensitivity.">Eosinophil activation and T lymphocyte infiltration in allergen-induced late phase skin reactions and classical delayed-type hypersensitivity.</a> J Immunol. 147, 816-822 (1991).</li><li>Flugel, A., Odoardi, F., Nosov, M., Kawakami, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoaggressive+effector+T+cells+in+the+course+of+experimental+autoimmune+encephalomyelitis+visualized+in+the+light+of+two-photon+microscopy.">Autoaggressive effector T cells in the course of experimental autoimmune encephalomyelitis visualized in the light of two-photon microscopy.</a> J Neuroimmunol. 191, 86-97 (2007).</li><li>Kawakami, N., 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=Live+imaging+of+effector+cell+trafficking+and+autoantigen+recognition+within+the+unfolding+autoimmune+encephalomyelitis+lesion.">Live imaging of effector cell trafficking and autoantigen recognition within the unfolding autoimmune encephalomyelitis lesion.</a> J Exp Med. 201, 1805-1814 (2005).</li><li>Miller, M. J., Wei, S. H., Parker, I., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+lymphocyte+motility+and+antigen+response+in+intact+lymph+node.">Two-photon imaging of lymphocyte motility and antigen response in intact lymph node.</a> Science. 296, 1869-1873 (2002).</li><li>Matheu, M. P. B., A, C. G. a. r. c. i. a., Chi, V., Rangaraju, S., Safrina, O., Monaghan, K., Uemura, M. I., Li, D., Pal, S., Monuki, E., Flugel, A., Pennington, M. W., Parker, I., Chandy, G. K., Calahan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Situ+Imaging+of+Effector/Memory+T+Cells+During+DTH+and+Suppression+by+Kv1.3+Channel+Block.">In Situ Imaging of Effector/Memory T Cells During DTH and Suppression by Kv1.3 Channel Block.</a> Immunity. , (2008).</li></ol>

imaging effector memory t cells in the ear after induction of adoptive dth

エフェクター/メモリーTリンパ球-遅延型過敏症（DTH）は、メインプレイヤーがCCR7される免疫反応です。ここで、我々は、ラットの耳にDTH反応の進行を誘導し、記録するための方法を示しています。エフェクター/メモリーT細胞応答-これは、CCR7の二光子励起イメージング用ラットの耳組織の準備のデモが続いている。 エフェクター/メモリーT細胞株（ビートン、C J.可視化実験、第8号） -養子DTHは、GFPで標識したOVA特異的CCR7の腹腔内注射により誘導される。次に、細胞を可視化を可能にするためにテキサスレッドの卵子と卵子共役の1:1混合（OVA - TR）と生理食塩水（対照耳）ともう片方の耳を注入することにより、チャレンジの48時間前のためにラットで平衡化されている常駐抗原提示細胞の。 我々は第二高調波発生によるコラーゲン線維の可視化と組み合わせて、イメージングのための有用な組織調製の方法免疫応答の間に深い真皮層内の細胞の運動性を説明。耳の組織は、5 × 5mmの正方形（少し大きめが良い）に切断し、その後37℃でのイメージングチャンバー内にシリコーングリースを使用して保護し、酸素バブリング組織培養培地で灌流されているVetbond™を使用してプラスチック製のカバースリップ、上にマウントされています° C 。

Watch this Scientific Journal Video about Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH at JoVE.com

Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH

ここでは、ラットの耳で遅延型-過敏（DTH）反応の進行を誘導し、記録するための方法を示しています。これは、エフェクター/メモリーT細胞応答の二光子イメージング用ラットの耳組織の準備のデモが続いている。

養子DTHの誘導後の耳のイメージングエフェクターメモリーT細胞

Delayed type hypersensitivity (DTH) is an immune reaction in which the main players are CCR7- effector / memory T lymphocytes. Here, we demonstrate a ...

imaging-effector-memory-t-cells-ear-after-induction-adoptive

Research

JoVE Journal

Biology

10.2K ビュー.  University of California, Irvine (UCI). ここでは、ラットの耳で遅延型-過敏（DTH）反応の進行を誘導し、記録するための方法を示しています。これは、エフェクター/メモリーT細胞応答の二光子イメージング用ラットの耳組織の準備のデモが続いている。

ビデオ: Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH

Induction and Monitoring of Active Delayed Type Hypersensitivity (DTH) in Rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of an antigen against which the immune system has been primed. The inflammatory reaction is characterized by redness and swelling of the site of antigenic challenge. It is a convenient model to determine the in vivo efficacy of immunosuppressants. Cutaneous DTH can be induced either by adoptive transfer of antigen-specific T lymphocytes or by active immunization with an antigen, and subsequent intradermal challenge with the antigen to induce the inflammatory reaction in a given skin area. DTH responses can be induced to various antigens, for example ovalbumin, tuberculin, tetanus toxoid, or keyhole limpet hemocyanin (KLH).
Here we demonstrate how to induce an active DTH reaction in Lewis rats. We will first prepare a water-in-oil emulsion of KLH, our antigen of interest, in complete Freund's adjuvant and inject this emulsion subcutaneously to rats. This will prime the immune system to develop memory T cells directed to KLH. Seven days later we will challenge the rats intradermally on the back with KLH on one side and with ovalbumin, an irrelevant antigen, on the other side. The inflammatory reaction will be visible 16-72 hours later and the red and swollen area will be measured as an indication of DTH severity.

Induction and Monitoring of Active Delayed Type Hypersensitivity DTH in Rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes. Here we demonstrate how to induce active DTH in Lewis rats and monitor the inflammatory response.

ラットにおけるアクティブ遅延型過敏（DTH）の誘導とモニタリング

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of ...

生物学

Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of an antigen against which the immune system has been primed. The inflammatory reaction is characterized by redness and swelling of the site of antigenic challenge. It is a convenient model to determine the in vivo efficacy of immunosuppressants. Cutaneous DTH can be induced either by adoptive transfer of antigen-specific T lymphocytes or by active immunization with an antigen, and subsequent intradermal challenge with the antigen to induce the inflammatory reaction in a given skin area. DTH responses can be induced to various antigens, for example ovalbumin, tuberculin, tetanus toxoid, or keyhole limpet hemocyanin. Such reactions can also be induced against autoantigen, for example to myelin basic protein (MBP) in rats with experimental autoimmune encephalomyelitis induced with MBP, an animal model for multiple sclerosis (1).
Here we demonstrate how to induce an adoptive DTH reaction in Lewis rats. We will first stimulate ovalbumin-specific T cells in vitro and inject these activated cells intraperitoneally to naive rats. After allowing the cells to equilibrate in vivo for 2 days, we will challenge the rats with ovalbumin in the pinna of one ear, while the other ear wil receive saline. The inflammatory reaction will be visible 3-72 hours later and ear thickness will be measured as an indication of DTH severity.

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T (TEM) lymphocytes. Here we demonstrate how to activate antigen-specific TEM cells, induce adoptive DTH in Lewis rats and monitor the inflammatory response.

ラットにおける養子遅延型過敏症の誘導とモニタリング

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

初代培養およびコルチのマウス臓器のプラスミドエレクトロ。

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Clonogenic Assay: Adherent Cells

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

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

クローン原性アッセイ：付着細胞

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

Imaging of Estrogen Receptor-&alpha; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen receptor -&alpha; and &beta;),estrogen receptor-&alpha; has been identified in both the smooth muscle and in endothelial cells of pial arterial segments using fluorescent staining combined with confocal laser scanning microscopy 4. Furthermore, ER-&alpha; is located in the nuclei and in the cytoplasm of rat basilar arteries 5. The receptors are abundant and fluoresce brightly, but clear visualization of discrete groups of receptors is difficult likely due to the numbers located in many cell layers of pial vessel segments. Additionally, many reports using immunohistochemical techniques paired with confocal microscopy poorly detail the requirements critical for reproduction of experiments 6. Our purpose for this article is to describe a simple technique to optimize the staining and visualization of ER-&alpha; using cross-sectional slices of pial arterioles obtain from female rat brains. We first perfuse rats with Evans blue dye to easily identify surface pial arteries which we isolate under a dissecting microscope. Use of a cryostat to slice 8 &mu;m cross sections of the arteries allows us to obtain thin vessel sections so that different vessel planes are more clearly visualized. Cutting across the vessel rather than use of a small vessel segment has the advantage of easier viewing of the endothelial and smooth muscle layers. In addition, use of a digital immunofluorescent microscope with extended depth software produces clear images of ten to twelve different vessel planes and is less costly than use of a confocal laser scanning microscope.

Imaging of Estrogen Receptor-&amp;#945; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

The goal of this article is to demonstrate a method to optimize immunofluorescent detection of estrogen receptor-&#945; (ER&#945;) in rat pial arterial slices using a digital immunofluorescent microscope.

デジタル免疫蛍光顕微鏡を用いたラット軟膜細動脈におけるエストロゲン受容体-αのイメージング

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen ...

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 ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

イメージングローカルCA<sup&gt; 2+</sup&gt;培養哺乳動物細胞内シグナル

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

リビングヒト臍帯静脈内皮細胞における高解像度タイムラプスイメージングと微小管ダイナミクスの自動解析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

動的細胞突起におけるソフトウェア支援トラッキングのためのグラフィカルユーザインタフェース

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

一次マウス網膜色素上皮細胞の効率的解離と培養

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...