ソリューション<ul><li>神経節細胞の細胞内溶液（mMで）：100 K -グルコン酸、8のKCl、1のMgCl 2、1 EGTA、10 HEPES、4 ATP、0.5 GTP、90μMスルホローダミンBの（染色用）、でpH = 7.4に調整KOH </li><li> 90 K -グルコン酸、8のKCl、1のMgCl 2、10 BAPTA、10 HEPES、4 ATP、0.5でpH = 7.4に調整GTP、90μMスルホローダミンB（染色用）、：双極細胞（mMで）のための細胞内液KOH </li><li>細胞外リンゲル溶液（mMで）：112塩化ナトリウム、5グルコース、5 HEPES、NaOHでpH = 7.75に調整2のKCl、2のCaCl 2、1のMgCl 2、。ソリューションの酸素化は必要ありません。 </li></ul>録音室を準備する私たちの記録室は、よく別の接着剤から切り出された小さなシリコンブロックは組織切片のために安定性を提供するために配置されるだけでなく接着剤付き顕微鏡のスライドで構成されています。 網膜スライスの準備光受容体の漂白を避けるために、郭清は、IRまたは暗赤色灯の光で行われる。一方が棒の経路からの応答を記録に興味を持っている場合は、唯一の赤外光は、（IRは、顕微鏡となく、部屋の明かりを解剖。追加の赤外線ゴーグルが顕微鏡下で行われない手続きのために使用される）を使用する必要があります。一、主に記録のコーンの光の応答に興味を持っている場合は、薄暗い赤い部屋の光または赤色閃光灯を使用することができます。顕微鏡は、しかし、それでもIR接眼レンズが必要です。 光受容体の入力が実験には無関係である場合、プロシージャは通常の顕微鏡を使用して、通常の室内光で行うことができる。 <ol><li>よく接着剤、シリコーンを持つ2つの顕微鏡スライドを準備してください（フィルタをプッシュ各スライド上にシリコーングリースの二つの平行なバンド（約0.5センチメートル離れて）を作成し、スライドの片側にフィルター長方形の紙を置いて、両方のグリースバンド交差それがうまくスライドにアタッチされるようにカミソリの刃の部分と紙） </li><li>動物はその後、断頭し、ダブルpithed、30分間氷浴中に置かれている：確立されたプロトコルに従ってサンショウウオを生け贄に捧げる。 </li><li>サンショウウオの頭から目を削除し、氷冷リンゲル液で湿らせたティッシュの紙の上に解剖顕微鏡下に置く</li><li>目から結合組織を取り除く</li><li>角膜に小さなカットをするためにカミソリの刃を使用して、 </li><li>目にその添付ファイルに沿ってハサミでそれを除去しながら鉗子で角膜を切るホールド</li><li>鉗子でレンズを取り外す</li><li>鉗子で虹彩を持ち、虹彩を削除するには、鋸状縁に沿って切断</li><li>二つの長方形の部分にアイカップを切る</li><li> （非常に慎重に押し下げながら、ピースを平らに確認してください）​​作品の一つをピックアップし、ろ紙上強膜側を上に置きます</li><li>静かに今ファイラ紙に添付すべき、網膜から強膜を持ち上げ</li><li>すぐに良くするために、氷冷リンゲル液を追加する</li><li>番目の部分に同じことを繰り返します。 </li><li> 200 -300μmの広いスライスをカットする特注のスライサーに配置されたカミソリの刃を使用してください。 </li><li>フィルター紙の側でスライスをピックアップしてピンセットを使用して、網膜の層が見えるようになるので、それを裏返し、真空グリースバンドの上に置きます</li><li>無傷に見える一は、いくつかのレイヤーを認識できる場合（倍率が充分ではないとして層は解剖顕微鏡下で確認することが困難である、しかし、スライスは通常、十分である網膜の層を示す水平バンドを表示するスライスを選択してください。それは）正立顕微鏡下で観察されると、スライスを使用するかどうかの最終決定は、しかし、行われる。つのスライドの間に呼び出し音のブリッジを構築することにより、記録のチャンバースライドに移動</li><li>スライスが傾いていないようにろ紙が録音室でゴムブロックに押し付けていることを確認してください（神経節細胞と光受容器が同時に焦点が合うはず） </li></ol>レコーディング機器で組織を配置する<ol><li>記録のスライドは、顕微鏡下に置き、重力の灌流systeを通じてリンゲルソリューションで灌流され</li><li>準備は10倍と40倍の水浸対物レンズと正立顕微鏡で可視化です。 </li><li>再び、我々は唯一の光受容体の漂白を防ぐために赤外光を使用してください。赤外線カメラは、テレビ画面に接続されている顕微鏡のサイドポートに接続されている。 </li></ol>視覚刺激視覚刺激はPsychtoolboxを使用して、MATLABでプログラミングされており、顕微鏡画像インジェクタ（MBFバイオサイエンス）を用いて顕微鏡の最上位ポートを介して網膜上に投影されています。ビット+ +デジタルビデオプロセッサ（ケンブリッジリサーチシステム）は、14ビット輝度のスケールを取得してsynchrにするために使用されますデータ収集と刺激プレゼンテーションをonize。このビデオで使用される視覚刺激は、安定した一様な背景（上2秒のために提示された100％のコントラストの明るいまたは暗いバー（幅=460μm）であり、輝度= 8 * 10 4光子/μm/ sの、サイズ：1.84 X 1.38ミリメートル）。 電気生理学<ul><li>マイクロピペットプラーで：パッチ電極は、ホウケイ酸ガラス（1.5mmのID 84ミリメ​​ートル。。OD）から抜き出されます。神経節細胞の場合、抵抗値は2〜4 Mohmをです。双極細胞の場合、4と8 Mohmを間に高い抵抗値が使用されます。 </li><li>電圧クランプ記録はMulticlamp 700A（Molecular Devices社）、アンプとLabVIEWでプログラミングされた研究室内部のソフトウェアを使用して実行されます。 </li><li>薬は、8チャンネルの灌流システムを介して灌流されています。 microperfusion電極は、スライス（あなたが血流をオンとオフ、1つのニーズが、プレッシャーはスライスがろ紙から切り離さになることができるように強すぎではないことに注意するときに組織が目に見えて移動する必要があります）の近くに配置されています。実験中にmicroperfusionは絶えず記録は血流のオンとオフを切り替えることによって邪魔されないように（何の薬が必要ない場合は、コントロールのソリューションを灌流する）オンにする必要があります。 </li></ul>

<ol>
	<li>Cook, P. B., Lukasiewicz, P. D., McReynnolds, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA(C)+receptors+control+adaptive+changes+in+a+glycinergic+inhibitory+pathway+in+salamander+retina.">GABA(C) receptors control adaptive changes in a glycinergic inhibitory pathway in salamander retina.</a> Journal of Neuroscience. 20, 806-812 (2000).</li><li>Heflin, S. J., Cook, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Narrow+and+wide+field+amacrine+cells+fire+action+potentials+in+response+to+depolarization+and+light+stimulation.">Narrow and wide field amacrine cells fire action potentials in response to depolarization and light stimulation.</a> Visual Neuroscience. 24, 197-206 (2007).</li><li>Ichinose, T., Shields, C. R., Lukasiewicz, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+Channels+in+the+Transient+Retinal+Bipolar+Cells+Enhance+Visual+Responses+in+Ganglion+Cells.">Sodium Channels in the Transient Retinal Bipolar Cells Enhance Visual Responses in Ganglion Cells.</a> J Neurosci.. 25, 1856-1865 (2005).</li></ol>

利点： <ol><li>すべての細胞型は、アクセス可能です </li><li>細胞型の簡単な識別は、特に、蛍光色素は、電極の溶液に添加されている場合 </li><li>薬理学的作用物質は、容易に標的細胞に到達することができます </li><li>パッチクランプ法は、網膜の計算では異なるイオンチャネルの役割を調査することができます。同じ情報が鋭い電極と細胞外スパイクの録音または録画を介して取得することが...

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		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Multiclamp 700A</td>
<td>Patch Clamp Amplifier</td>
<td>Molecular Devices</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>SZX9</td>
<td>Dissection Micropscope</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>BX50WI</td>
<td>Upright Microscope</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>Equipped with 40x, 10x water immersion objective, fluorescent filters and mercury lamp</td>
</tr>
<tr>
<td>P-97</td>
<td>Micropipette Puller</td>
<td>Sutter Instruments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Lucivid</td>
<td>Microscope image injector</td>
<td>Mbf bioscience</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>8-channel perfusion system</td>
<td>Microperfusion</td>
<td>Parker Hannfin corporation</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Bits++</td>
<td>Digital Video Processor</td>
<td>Cambridge Research Systems</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Infrared Oculars</td>
<td>Other</td>
<td>ITT</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Adhesive silicone wells</td>
<td>Other</td>
<td>Molecular Probes</td>
<td>&nbsp;</td>
<td>20mm diameter, 1.0mm deep</td>
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<tr>
<td>Membrane Filters</td>
<td>Filter</td>
<td>Milipore</td>
<td>HAWPO1300</td>
<td>.45&#181;m HA</td>
</tr>
<tr>
<td>Borosilicate Glass Capillaries</td>
<td>Electrode glass</td>
<td>World Precision Instruments</td>
<td>1B150-4</td>
<td>&nbsp;</td>
<tr>
<td>Syringe Filters</td>
<td>Filter</td>
<td>Whatman</td>
<td>6789-0402</td>
<td>4mm filters, .2um Nylon Membrane, Polypropylene housing</td>
</tr>
<tr>
<td>IR camera</td>
<td>Micropscope mounted camera</td>
<td>Sony</td>
<td>SPT-M324</td>
<td>Extrawave HAD, B7W video camera</td>
</tr>
<tr>
<td>Picrotoxin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P1675</td>
<td>&nbsp;</td>
<tr>
<td>Strychnine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S0532</td>
<td>&nbsp;</td>
<tr>
<td>Imidazole-4-acetic acid sodium salt</td>
<td>Reagent</td>
<td>Sigma</td>
<td>I7013</td>
<td>&nbsp;</td>
<tr>
<td>Larval tiger salamanders</td>
<td>Animal</td>
<td>Charles E. Sullivan (Nashville, TN)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
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whole-cell recordings of light evoked excitatory synaptic currents in the retinal slice

我々は、シナプス回路が網膜の基礎となる視覚情報の処理を研究するためにホールセルパッチクランプ法を使用してください。このビデオでは、我々は、網膜のスライス標本における個々のセルの光誘発電流の全細胞記録を実行するプロセスをガイドします。我々は、動物モデルとして水生トラフサンショウウオを使用してください。我々は目の解剖を記述することで開始し、スライスを電気生理学的記録のためにマウントする方法を示します。スライスが録音室に配置されると、我々は、ホールセル電圧クランプ記録を実行する方法を示します。我々は、その後、光誘発電流応答を引き出すためにスライスに光受容体の上に視覚刺激を投影。録音中に我々は、8チャンネルの灌流システムは、私たちはすぐに別のエージェントを切り替えることができることにより、薬理学的薬剤とスライスを灌流。網膜スライス標本は広く、特にホールマウントの準備にはアクセスできないアマクリンまたは双​​極細胞を、研究するために、網膜でのパッチクランプ記録のために使用されます。

Watch this Scientific Journal Video about Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice at JoVE.com

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

このビデオでは、水生トラフサンショウウオの網膜スライスにおけるホールセル電圧クランプ記録のプロセスを示しています。網膜の視覚刺激時のパッチクランプ記録を実行する方法を我々は、だけでなく、スライスの準備を示しています。

光の全細胞録音は網膜スライスにおける興奮性シナプス電流を誘発

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we ...

whole-cell-recordings-light-evoked-excitatory-synaptic-currents

Research

JoVE Journal

Biology

12.7K ビュー.  Boston University. このビデオでは、水生トラフサンショウウオの網膜スライスにおけるホールセル電圧クランプ記録のプロセスを示しています。網膜の視覚刺激時のパッチクランプ記録を実行する方法を我々は、だけでなく、スライスの準備を示しています。

ビデオ: Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

Horizontal Slice Preparation of the Retina

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of retinal neurons, such as amacrine cells, expand their dendrites horizontally, so that the morphology of the cells is supposed to be severely damaged in the vertical slices. In the whole-mount preparation, especially for patch-clamp recordings, retinal neurons in the middle layer are not easily accessible due to the extensive coverage of glial cell (Mueller cell) s endfeets. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact. The slice was made horizontally at the inner layer of the retina using a vibratome slicer after the retina was embedded in the low-temperature melting agarose gel. In this horizontal slice preparation of the retina, we studied the function of retinal neurons compared with their morphology, by using patch-clamp recording, calcium imaging technique, immunocytochemistry, and single-cell RT-PCR.

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact.

網膜の水平スライス標本

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of ...

生物学

Whole Cell Recordings from Brain of Adult Drosophila

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by describing the dissecting solution and capture of the adult females used in our studies. The procedure for removing the whole brain intact, including both optic lobes, is illustrated. Dissection of the overlying trachea is also shown.  The isolated brain is not only small but needs special care in handling at this stage to prevent damage to the neurons, many of which are close to the outer surface of the tissue. We show how a special holder we developed is used to stabilize the brain in the recording chamber. A standard electrophysiology set up is used for recording from single neurons or pairs of neurons. A fluorescent image, viewed through the recording microscope, from a GAL4 line driving GFP expression (GH146) illustrates how projection neurons (PNs) are identified in the live brain. A high power Nomarski image shows a view of a single neuron that is being targeted for whole cell recording. When the brain is successfully removed without damage, the majority of the neurons are spontaneously active, firing action potentials and/or exhibiting spontaneous synaptic input. This in situ preparation, in which whole cell recording of identified neurons in the whole brain can be combined with genetic and pharmacological manipulations, is a useful model for exploring cellular physiology and plasticity in the adult CNS.

This video demonstrates the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons using standard whole cell technology. It includes images of GFP labeled cells and neurons viewed during recording.

アダルトショウジョウバエの脳から全細胞レコーディング

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by ...

Major Components of the Light Microscope

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

光学顕微鏡の主要なコンポーネント

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for ...

Recordings of Neural Circuit Activation in Freely Behaving Animals

The relationship between patterns of neural activity and corresponding behavioral expression is difficult to establish in unrestrained animals. Traditional non-invasive methods require at least partially restrained research subjects, and they only allow identification of large numbers of simultaneously activated neurons. On the other hand, small ensembles of neurons or individual neurons can only be measured using single-cell recordings obtained from largely reduced preparations. Since the expression of natural behavior is limited in restrained and dissected animals, the underlying neural mechanisms that control such behavior are difficult to identify. 
Here, I present a non-invasive physiological technique that allows measuring neural circuit activation in freely behaving animals. Using a pair of wire electrodes inside a water-filled chamber, the bath electrodes record neural and muscular field potentials generated by juvenile crayfish during natural or experimentally evoked escape responses. The primary escape responses of crayfish are mediated by three different types of tail-flips which move the animals away from the point of stimulation. Each type of tail-flip is controlled by its own neural circuit; the two fastest and most powerful escape responses require activation of different sets of large &#8220;command&#8221; neurons. In combination with behavioral observations, the bath electrode recordings allow unambiguous identification of these neurons and the associated neural circuits. Thus activity of neural circuitry underlying naturally occurring behavior can be measured in unrestrained animals and in different behavioral contexts.

Non-invasive measurements of neural activity patterns in freely behaving animals are obtained by combining neurophysiological recordings with high speed videography.

自由に動物を振る舞うの神経回路のアクティベーションの記録

The relationship between patterns of neural activity and corresponding behavioral expression is difficult to establish in unrestrained animals. ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

マウスの錐体光受容体からの単一細胞サクション録音

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

Whole-Cell Recording of Calcium Release-Activated Calcium (CRAC) Currents in Human T Lymphocytes

In T lymphocytes, depletion of Ca2+ from the intracellular Ca2+ store leads to activation of plasmalemmal Ca2+ channels, called Calcium Release-Activated Calcium (CRAC) channels. CRAC channels play important role in regulation of T cell proliferation and gene expression. Abnormal CRAC channel function in T cells has been linked to severe combined immunodeficiency and autoimmune diseases 1, 2 . Studying CRAC channel function in human T cells may uncover new molecular mechanisms regulating normal immune responses and unravel the causes of related human diseases. Electrophysiological recordings of membrane currents provide the most accurate assessment of functional channel properties and their regulation. Electrophysiological assessment of CRAC channel currents in Jurkat T cells, a human leukemia T cell line, was first performed more than 20 years ago 3, however, CRAC current measurements in normal human T cells remains a challenging task. The difficulties in recording CRAC channel currents in normal T cells are compounded by the fact that blood-derived T lymphocytes are much smaller in size than Jurkat T cells and, therefore, the endogenous whole-cell CRAC currents are very low in amplitude. Here, we give a step-by-step procedure that we routinely use to record the Ca2+ or Na+ currents via CRAC channels in resting human T cells isolated from the peripheral blood of healthy volunteers. The method described here was adopted from the procedures used for recording the CRAC currents in Jurkat T cells and activated human T cells 4-8.

Whole-Cell Recording of Calcium Release-Activated Calcium CRAC Currents in Human T Lymphocytes

We provide a step-by-step protocol for whole-cell patch clamp recording of Calcium Release-Activated Calcium (CRAC) currents in peripheral blood mononuclear cell-derived human T lymphocytes.

ヒトTリンパ球におけるカルシウム放出、活性化カルシウム（CRAC）電流のホールセル記録

In T lymphocytes, depletion of Ca2+ from the intracellular Ca2+ store leads to activation of plasmalemmal Ca2+ channels, called Calcium Release-Activated ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

全細胞集団の単一細胞レベルでサイクリンBの分解を勉強

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

外植片培養での歯胚の開発に続いてのためのスライス培養法

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

成体マウス頭蓋冠骨髄中の造血幹細胞および前駆細胞のin vivo 4次元トラッキングで

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

酸化ストレスに赤/近赤外光療法の波長および強度の範囲の効果の評価のための手法<em&gt;インビトロ</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...