我々は、この原稿の重要な読書のために松浪研究室のメンバーに感謝。

1。手順： 転送細胞、トランスフェクションおよび免疫細胞化学、日ごとに行わつのステップ：プロシージャでは、3日間の合計時間をかけて完成した三つの部分から構成されています。最初の二日間での転送と細胞のトランスフェクションは、層流室内の無菌状態で実施する必要があります。 1日目：表面発現アッセイのためのHEK293T細胞の譲渡。 <ol><li>伝送媒体（M10）：ミックス10％（体積）無菌状態のウシ胎児血清を添加した最小必須培地。 </li><li>セットアップに必要なプレートの数に応じて100mmの培養プレート内の目的のコンフルエントに細胞を成長させる。それは草に覆わまたは疎セルを避けるために転送されるセルの割合を決定することが重要である：例えば、100％コンフルエントな100 mmディッシュから、一つでコンフルエントに約30％を取得するために35mm皿に約3％を譲渡することができます。後者。 </li><li>前に無菌層流室、コート22に、セルを転送する滅菌ポリDリジン（1mgの/ mL）と各35 mmの細胞培養皿で片足、メッキの細胞のためのコーティング面を上X 22 mmのガラスカバースリップ（図1 ）。 10分 - 5のために乾燥してソリューションを許可する。必要に応じてこの間隔の間に、層流室内のUV光源は、カバースリップを滅菌するためにオンにすることができます。ポリDリジンは、異種細胞がカバーガラスに付着することができます。 </li><li> 100mmディッシュ内の既存のメディアアウト吸引、細胞を転送するには、慎重に8 mLの滅菌PBSを添加し洗浄、吸引PBS、3 mLの0.05％トリプシン- EDTAを用いて細胞をトリプシン処理、細胞が培養皿からデタッチするときに、によって、さらに酵素反応をブロックする5 mLのM10を追加。細胞をひいて粉にすると無菌コニカルチューブに必要なボリュームを移転すること、細胞をペレットに5分間200グラムで遠心。無傷の細胞のペレットを残し、トリプシン- EDTAを含有する上清を吸引除去する。 35mmの皿に細胞を転送するには、それぞれの料理のために1 mLのM10を追加し、細胞を解離する粉薬。各ディッシュに再懸濁した細胞を1 mLを追加。 37℃で一晩培養する° C、5％CO 2。 <img src="/files/ftp_upload/2405/2405fig1.jpg" alt="図1" /> 図1コート22ポリDリジンでX 22 mmのガラス製カバースリップと各35 mmのシャーレに1コーティング面を上に置きます。 </li></ol> 2日目：HEK293T細胞のトランスフェクション。 <ol><li> 18-24時間転送した後、細胞は、アッセイする受容体のトランスフェクションのための準備が整いました。 4の前に説明したキアゲンミニプレップキット以下のプロトコルを使用して細菌培養液から調製した哺乳動物発現ベクターpCI、でクローニングされた受容体とRTP1Sを使用してください。 N末端に免疫染色を容易にするために20アミノ酸の長ロドプシンエピトープでタグ付けされた受容体を使用してください。 </li><li>受容体構造、RTP1S構造と緑色蛍光タンパク質それぞれのトランスフェクションの50 ngの250 ngの1000ngを使用してください。メーカーのマニュアルに従ってリポフェクタミン2000を、使用してトランスフェクションを実行します。 </li></ol> 3日目：細胞表面の染色された受容体を可視化する免疫細胞。 <ol><li>開始する前に、染色やソリューションを洗濯を準備し、4℃に冷却する。培地染色：最小必須培地、45mlの、ウシ胎児血清、5 mLを、HEPES（1M）、500μL（最終濃度10mMの）、アジ化ナトリウム（1.5M）、500μL（最終濃度15MM）。 、HEPES（1M）、5mLの（最終濃度10mMの）、アジ化ナトリウム（1.5M）、5mLの（最終濃度15mm）をHBSS、500mLの：ソリューションを洗ってください。どちらのソリューションは、再利用のために4℃で保存することができます。染色媒体100倍で、必要な抗体を希釈することにより抗体の溶液を調製。 </li><li> 24時間後にトランスフェクションについての嗅覚受容体のための表面染色のための免疫化学を開始します。生細胞の染色を実行するには、実験的なセットアップが4に冷却しなければならない° C：氷とパットのようにもできる限りそれを作るために氷の表面に含まれる大を埋める、氷の上にトレイを置き、70％でそれを吹きかけエタノール。滑らかな表面を得るために、パラフィルムの部分（トレイの長さよりも小さい）とエタノールスプレートレイ上に貼ってカット。ピンセットを使用して、パラフィルム上にトランスフェクトされた細胞、一度、セル側を含むガラスカバースリップを転送します。層ごとにカバーはスリップ（図2）のコーナーから慎重に押し出し、100μLコールド主染色液でスリップ。階層化抗体溶液を持つすべてのカバースリップの後、空気中のソリューションの枯渇を防ぐためにトレイをカバーしています。 45〜60分間一次インキュベーションを行う。 </li><li>主なインキュベーションの後、すぐにメディアを含む、オリジナルの35mm培養皿にカバースリップを転送する。培地を吸引除去し、カバースリップから細胞を失うことを防ぐために皿の縁から静かに2 mLの冷却した洗浄溶液を加える。洗浄液を吸引除去し、3回の洗浄を完了するために二度繰り返す。第三洗浄の終了時に、洗浄液を吸引しないでください。 </li><li>二次抗体solutiを準備染色のメディアで上と同じトレイに新しく貼らパラフィルム層に洗浄溶液からカバースリップを転送する。再び静かに一次インキュベーションなどのコーナーから、層ごとにカバースリップをに抗体溶液100μLを押し出します。 30の二次インキュベーションを行う - 45分とオリジナルの35mmの皿の洗浄溶液に戻って転送する。もう一度、カバーが説明されているように三回伝票は洗浄。 </li><li>最後の洗浄からソリューションを洗浄し、2 mLの1パーセントチルドパラホルムアルデヒドを追加吸引する。 15分間氷上で、チルド1％パラホルムアルデヒド溶液で細胞を固定する。 </li><li>きれいな顕微鏡のガラスのカバースリップがダウンしてセルの側で、マウント試薬のMowiolを使用してスライドし、慎重にすべての気泡を（図3）除外マウントします。かつてMowiol空気が乾燥する、表面に蒸留水を加えるとすぐに吸引し、静かにカバースリップを洗う。 <img src="/files/ftp_upload/2405/2405fig2.jpg" alt="図2" /> 図2。レイヤ100μLの冷主要な染色液でそれぞれカバー。 <img src="/files/ftp_upload/2405/2405fig3.jpg" alt="図3" /> 図3。Mowiolマウンティング試薬を使用してスライドするきれいなガラスの顕微鏡で下のセル側にカバースリップをマウントします。 </li></ol> 2。代表的な結果： 生細胞の染色は1つがシャペロン（プロテインRTP1Sを輸送受容体とこのケースの嗅覚受容体Olfr62、の）（図4）と受容体のトランスフェクションで、細胞の表面上のタンパク質を可視化することができます。受容体の細胞表面の染色は、染色の点状のパターン（図4B、挿入図）によって特徴付けられる。不適切に行わ染色することが困難オブザーバーは、ノイズから実際の表面染色を区別するためになり、より高いノイズにつながる可能性があります。 <img src="/files/ftp_upload/2405/2405fig4.jpg" alt="図4" /> 図4。シャペロンと受容体のトランスフェクションは、生細胞の染色を用いて細胞の表面上のタンパク質の可視化が可能になります。嗅覚だけでは受容体Olfr62（）とRTP1Sシャペロン（B）とOlfr62のトランスフェクションのトランスフェクション。 GFPの発現レベルは、トランスフェクションのコントロール（A&#39;とB&#39;）として機能します。

<ol>
	<li>Buck, L., Axel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+multigene+family+may+encode+odorant+receptors:+a+molecular+basis+for+odor+recognition.">A novel multigene family may encode odorant receptors: a molecular basis for odor recognition.</a> Cell. 65, 175-187 (1991).</li><li>Malnic, B., Hirono, J., Sato, T., Buck, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+receptor+codes+for+odors.">Combinatorial receptor codes for odors.</a> Cell. 96, 713-723 (1999).</li><li>Saito, H., Kubota, M., Roberts, R. W., Chi, Q., Matsunami, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RTP+family+members+induce+functional+expression+of+mammalian+odorant+receptors.">RTP family members induce functional expression of mammalian odorant receptors.</a> Cell. 119, 679-691 (2004).</li><li>Zhuang, H., Matsunami, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+cell-surface+expression+and+measuring+activation+of+mammalian+odorant+receptors+in+heterologous+cells.">Evaluating cell-surface expression and measuring activation of mammalian odorant receptors in heterologous cells.</a> Nat Protoc. 3, 1402-1413 (2008).</li></ol>

利害の衝突は宣言されません。

各ステップは、著名な、独特の表面の汚れを確実にするために慎重に実施しなければならない。全体の染色工程は、（氷上で）寒さで実施しなければならない、とカバースリップが配置されているトレイは、細胞が生き続けることを確認するために使用する前に冷却する必要があります。また固定は表面染色のプロセスの最後に実行されます。これは固定では一次および二次抗体に細胞膜を?...

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<tr>
<td>Cover slip 22 X 22 mm (#1)</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>72200-11</td>
<td>&nbsp;</td>
<tr>
<td>Fetal bovine serum</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>16000</td>
<td>&nbsp;</td>
<tr>
<td>Lipofectamine 2000</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11668-019</td>
<td>&nbsp;</td>
<tr>
<td>Minimal essential medium</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M4655</td>
<td>With L glutamate, Earle&#x2019;s salt and bicarbonate</td>
</tr>
<tr>
<td>Mowiol</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>475904</td>
<td>&nbsp;</td>
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<td>Paraformaldehyde</td>
<td>&nbsp;</td>
<td>EMS</td>
<td>19208</td>
<td>&nbsp;</td>
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<td>Hanks Balanced Salt Solution (HBSS) 1X </td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>14025</td>
<td>With calcium chloride and magnesium chloride</td>
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<tr>
<td>HEPES Buffer Solution</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15630</td>
<td>&nbsp;</td>
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<td>Phosphate Buffer Saline (PBS)</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>21-040-CV</td>
<td>Without calcium and magnesium</td>
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<td>Poly D Lysine Hydrobromide</td>
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<td>Sigma</td>
<td>P7280</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>EM Science</td>
<td>SX0299-1</td>
<td>&nbsp;</td>
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<td>Tissue culture dish 35 X 10 mm</td>
<td>&nbsp;</td>
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<td>353801</td>
<td>&nbsp;</td>
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<td>Trypsin-EDTA </td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25300</td>
<td>0.05%</td>
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assaying surface expression of chemosensory receptors in heterologous cells

悪臭の鮮やかな世界は、嗅覚の感覚によって認識されています。マウスでの嗅覚は、Gタンパク質Gαolfと結合することによって揮発性の匂い分子を結合し、細胞内の信号に細胞外シグナルに変換するために仮定されている約1200のGタンパク質共役受容体（GPCR）1のレパートリーによって媒介される。受容体への臭気物質の結合を組み合わせルールに従うと考えられている、つまり、1つの嗅覚は、いくつかの受容体に結合する可能性があり、つの受容体は、程度の差は2に、複数の匂い物質を結合することができる。生化学、シグナリングおよびリガンド結合研究は、便利な異種細胞を使用するほとんどのGPCRのために行われている。トランスフェクションに彼らは表面にエクスポートに失敗したので、しかし、嗅覚受容体の研究のために異種細胞の使用は長い間排除されました。齋藤らは、家族のシャペロンを共が一緒に3をトランスフェクトされた異種細胞の表面へのトラフィックの嗅覚受容体へのシャペロンとしての嗅覚ニューロンと行為で強化された発現を示すタンパク質を輸送する単一の細胞膜の通過の受容体（RTP）を示している。受容体は細胞株における堅牢な表面発現を示す場合に異種細胞を使用し受容するための生化学的アッセイを行うには、最初に決定する必要があります。これは、生細胞の染色に続いてシャペロンのRTP1Sと受容体は、蛍光細胞外ドメインまたは排他的に細胞外ドメイン内のタグにラベルを付けるために過剰発現によってアッセイすることができます。ここでは、便利なHEK293T細胞の表面上の嗅覚受容体を検出するために使用することができる生細胞の染色を実施するためのプロトコルを示しています。さらに、それはまた他の化学感覚受容体やGPCRの表面発現のためのアッセイに使用することができます。

Watch this Scientific Journal Video about Assaying Surface Expression of Chemosensory Receptors in Heterologous Cells at JoVE.com

Assaying Surface Expression of Chemosensory Receptors in Heterologous Cells

ここでは、便利なHEK293T細胞の表面上の嗅覚受容体を検出するために使用することができる生細胞の染色を実施するためのプロトコルを示しています。さらに、それはまた他の化学感覚受容体やGPCRの表面発現のためのアッセイに使用することができます。

異種細胞での化学感覚受容体の表面発現をアッセイ

The vivid world of odors is recognized by the sense of olfaction. Olfaction in mice is mediated by a repertoire of about 1200 G Protein Coupled Receptors ...

assaying-surface-expression-chemosensory-receptors-heterologous

Research

JoVE Journal

Neuroscience

10.8K ビュー.  Duke University. ここでは、便利なHEK293T細胞の表面上の嗅覚受容体を検出するために使用することができる生細胞の染色を実施するためのプロトコルを示しています。さらに、それはまた他の化学感覚受容体やGPCRの表面発現のためのアッセイに使用することができます。

ビデオ: Assaying Surface Expression of Chemosensory Receptors in Heterologous Cells

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) pacemakers or clocks. Malfunctions in the human circadian system are associated with numerous diseases or disorders. Much progress towards our understanding of the mechanisms underlying circadian rhythms has emerged from genetic screens whereby an easily measured behavioral rhythm is used as a read-out of clock function. Studies using Drosophila have made seminal contributions to our understanding of the cellular and biochemical bases underlying circadian rhythms. The standard circadian behavioral read-out measured in Drosophila is locomotor activity. In general, the monitoring system involves specially designed devices that can measure the locomotor movement of Drosophila. These devices are housed in environmentally controlled incubators located in a darkroom and are based on using the interruption of a beam of infrared light to record the locomotor activity of individual flies contained inside small tubes. When measured over many days, Drosophila exhibit daily cycles of activity and inactivity, a behavioral rhythm that is governed by the animal's endogenous circadian system. The overall procedure has been simplified with the advent of commercially available locomotor activity monitoring devices and the development of software programs for data analysis. We use the system from Trikinetics Inc., which is the procedure described here and is currently the most popular system used worldwide. More recently, the same monitoring devices have been used to study sleep behavior in Drosophila. Because the daily wake-sleep cycles of many flies can be measured simultaneously and only 1 to 2 weeks worth of continuous locomotor activity data is usually sufficient, this system is ideal for large-scale screens to identify Drosophila manifesting altered circadian or sleep properties.

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

We describe procedures for recording daily locomotor activity rhythms of Drosophila and subsequent data analysis. Locomotor activity rhythms are a reliable behavioral output of animal circadian clocks and are used as the standard readout of clock function when studying circadian mutants or examining how the environment regulates the circadian system.

に概日リズムと睡眠のパラメータを研究するために運動活性を測定するショウジョウバエ</em

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) ...

神経科学

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of these diseases, including Alzheimer's, Parkinson's and Huntington's disease are characterized by age-dependent deterioration in learning and memory functions and movement coordination2. Here we use behavioral assays, including the negative geotaxis assay3 and the aversive phototaxic suppression assay (APS assay)4,5, to show that some of the behavior characteristics associated with human neurodegeneration can be recapitulated in flies. In the negative geotaxis assay, the natural tendency of flies to move against gravity when agitated is utilized to study genes or conditions that may hinder locomotor capacities. In the APS assay, the learning and memory functions are tested in positively-phototactic flies trained to associate light with aversive bitter taste and hence avoid this otherwise natural tendency to move toward light. Testing these trained flies 6 hours post-training is used to assess memory functions. Using these assays, the contribution of any genetic or environmental factors toward developing neurodegeneration can be easily studied in flies.

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Behavior assays for measuring locomotor functions, learning, and memory abilities in Drosophila.

でアッセイする運動、学習、記憶障害ショウジョウバエ</em神経変性の>モデル

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of ...

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

脊髄電気生理学II：細胞外吸引電極の作製

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

無傷の感覚器官の前駆細胞の生細胞イメージングショウジョウバエ蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

げっ歯類やヒトから鼻嗅覚幹細胞を単離する

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

開発神​​経上皮における細胞挙動の高解像度のライブイメージング

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.
	The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell 1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.

The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.

コメットアッセイを用いて海馬神経細胞のDNA損傷を測定する

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons responsible for proprioception as they are detecting joint position and movement, and muscle tension. Electrical activity from crustacean proprioceptors and tension receptors is recorded by basic neurophysiological instrumentation, and a transducer is used to simultaneously measure force that is generated by stimulating a motor nerve. In addition, we demonstrate how to stain the neurons for a quick assessment of their anatomical arrangement or for permanent fixation. Staining reveals anatomical organization that is representative of chordotonal organs in most crustaceans. Comparing the tension nerve responses to the proprioceptive responses is an effective teaching tool in determining how these sensory neurons are defined functionally and how the anatomy is correlated to the function. Three staining techniques are presented allowing researchers and instructors to choose a method that is ideal for their laboratory.

Physiological and anatomical techniques are demonstrated to address function and structure for joint proprioceptors and muscle tension receptors in crustacean walking limbs.

カニ手足の固有感覚と緊張受容体：学生実験室の練習

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

神経発火率を操作することによってアストロサイト受容体の可塑性を誘導すること

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at the plasma membrane. Here, we present detailed protocols for 2 methods that facilitate the study of such processes, both of which are based on the principle of the biotinylation of cell-surface proteins. The first is designed to allow for the semi-quantitative determination of the relative levels of a particular protein at the cell-surface. In it, the lysine residues of the plasma membrane proteins of cells are first labeled with a biotin moiety. Once the cells are lysed, these proteins may then be specifically precipitated via the use of agarose-immobilized streptavidin by exploiting the natural affinity of the latter for biotin. The proteins isolated in such a manner may then be analyzed via a standard western blotting approach. The second method provides a means of determining the endocytic rate of a particular cell-surface target over a period of time. Cell-surface proteins are first modified with a biotin derivative containing a cleavable disulfide bond. The cells are then shifted back to normal culture conditions, which causes the endocytic uptake of a proportion of biotinylated proteins. Next, the disulfide bonds of non-internalized biotin groups are reduced using the membrane-impermeable reducing agent glutathione. Via this approach, endocytosed proteins may thus be isolated and quantified with a high degree of specificity.

Two biotinylation-based methods, designed for determining the cell-surface expression and endocytic rate of proteins expressed at the plasma membrane, are presented in this report.

ビオチン化を用いた初代星状細胞培養におけるタンパク質の細胞表面発現およびエンドサイトーシス速度の測定

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at ...