作者ありがとう淵チュ チョン彼女の実験的なサポート synaptosome 精製と Jungjoo 公園の中に PS 露出シナプトソームのイメージの。さらに、役に立つ議論のため鄭研究室のすべてのメンバーに感謝いたします。この作品は、韓国 (NRF) 交付金 (MSIP) (NRF 2016M3C7A1905391 と NRF 2016R1C1B3006969) の韓国の政府によって資金を供給の国立研究財団によって支えられた (W. S.C)。

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この記事では、長期ライブ イメージングの in vitro貪食能試金精製のグリア細胞と pH インジケーター共役シナプトソームを使用しての手法を提示します。ミクログリアと比較して示すアストロ サイト シナプトソーム貪食刺激による別の貪食・分解能力を持っています。さらに、我々 のデータは、架橋分子 GAS6、蛋白質、MEGE8 などが含まれて、アストロ サイト分泌の要因が効率的な PS ?...

脳の非興奮性細胞を参照、グリア細胞は、中枢神経系 (CNS) における主要な細胞タイプです。以前は、グリア細胞は、主に神経細胞の生存と基底のシナプス特性を維持するために受動の役割を果たす単なる支持細胞として見なされていた。ただし、出現の証拠は明らかにしたグリア細胞がシナプス形成1,2,3とシナプスを媒介する脳のホメオスタシス維持などの神経生物学のさまざまな側面でより積極的な役割を果たしています。除去4、5、および変調シナプス可塑性6,7。中枢神経系のグリア細胞には、オリゴデンドロ サイト、ミクログリア、アストロ サイトが含まれます。これらの細胞、アストロ サイト、ミクログリアの中でシナプス4,5、アポトーシス細胞8、ニューラル破片9、およびアミロイド β などの病原性タンパク質を巻き込むによる貪食の役割を果たすことが示されています。斑10,11。脳の発達、アストロ サイトから MERTK と神経: MEGF10 依存貪食4背側外側膝状核 (dLGN) でシナプスを排除します。同様に、ミクログリアはまた古典的な補体カスケード5を通じて発達段階のシナプスの C1q コーティングを除去します。興味深いことに、シナプス刈り込みの欠陥がいくつかの神経疾患の創始者の 1 つをすることができますが示唆されています。たとえば、(C4) に補体成分 4 の変異ミクログリアによって補体を介したシナプス刈り込みを生じ、強く人間12で統合失調症の有病率に関連付けられて示されています。最近の論文では、古典的補体経路 hyperactivated は、広告の開始段階とこの疾患13初期シナプス損失を誘発することも示しています。
ミクログリアを介した貪食と比べると、アストロ サイトを介した貪食が開始し、さまざまな神経疾患の進行に寄与するかどうかは不明瞭です。しかし、最近の論文は示唆やアストロ サイトによる通常のシナプス刈り込みの率を変える要素脳のホメオスタシスを混乱させる可能性があります広告感受性と病理14に貢献。アストロ サイトによるシナプス刈り込みの力強く、アポの異性、広告 (ApoE2) の保護の対立遺伝子と強く強化率と率を大幅に削減 (ApoE4) の広告の危険の対立遺伝子によって制御されます。また、 ApoE4を発現するトランスジェニック マウスの制御やApoE2マウス14よりもはるかに多くのシナプス C1q 蓄積。これらのデータは、初期のアストロ サイトを介した貪食能を障害する示唆している AD 脳活性化補体を介したミクログリア細胞貪食、シナプス変性を運転する老齢 C1q コーティング シナプス/シナプスの残骸の蓄積を引き起こす可能性があります.ApoE4キャリアにおけるアストロ サイトの障害の貪食能も広告の影響を受けた脳アミロイド ・ ベータ斑の制御不能な蓄積に貢献するかもしれない。
さらに、高齢者のショウジョウバエ脳のグリア細胞がドレイパー、アストロ サイトがシナプスを phagocytosing 用神経: Megf10の相同物の減らされた翻訳による貪食能力を失うことが示されています。ドレイパー レベルを復元救出効率的に瓦礫損傷した軸索が老化誘導を示す若い脳と同程度に高齢者の脳のグリア細胞の貪食能の貪食能の変化アストロ サイト脳のホメオスタシスの15の破壊に貢献するかもしれない。
これらの新しい調査結果に基づいて、予防し、様々 な脳神経疾患を治療する魅力的な治療戦略になる可能性がありますアストロ サイトの貪食能を調節すること。この点で、リソソーム酸性ナノ16の酸性化を誘導し、トランスクリプション要因 EB (TFEB) は、強化することができますを過剰発現によるアストロ サイトなどの貪食能を強化するいくつかの試みがずっとあります。リソソーム器官17。これらの試みにもかかわらずまだわかりにくい各アストロ サイトやミクログリア細胞の貪食反応速度論で異なると増加か各種疾患における貪食能力を減少する必要がありますか。
アストロ サイトの貪食能をリアルタイムで検出するための新規の in vitroアッセイを提案します。データは、アストロ サイト、ミクログリアの貪食・分解の別の速度を表示します。アストロ エアコン媒体 (ACM)、アストロ サイトから分泌因子が含まれ、アストロ サイト、ミクログリアの効果的な貪食能に不可欠です。さらに、神経: Megf10、アストロ サイト、 Ced 1とドレイパーの相同物の貪食受容体貪食のアストロ サイトを介した8,18で重要な役割を果たしています。

<table>
	<tbody>
		<tr>
			<td>Synaptosome purification</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>GE healthcare life sciences</td>
			<td>17-0891-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Quick Start Bradford Protein Assay Kit 2</td>
			<td>BIO-RAD</td>
			<td>5000202</td>
			<td></td>
		</tr>
		<tr>
			<td>pH indicator conjugation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide(DMSO)</td>
			<td>LPS solution</td>
			<td>DMSO100</td>
			<td></td>
		</tr>
		<tr>
			<td>pHrodo red, succinimidyl ester</td>
			<td>Molecula probes</td>
			<td>P36600</td>
			<td></td>
		</tr>
		<tr>
			<td>Immunopanning</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10X Earle&#8217;s balanced salt solution (EBSS)</td>
			<td>Sigma</td>
			<td>E7510</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Bovogen</td>
			<td>BSA025</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyrebonuclease 1 (DNase)</td>
			<td>Worthington</td>
			<td>Is002007</td>
			<td></td>
		</tr>
		<tr>
			<td>(DMEM)</td>
			<td>Gibco</td>
			<td>11960-044</td>
			<td></td>
		</tr>
		<tr>
			<td>(dPBS)</td>
			<td>Welgene</td>
			<td>LB001-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Griffonia Simplicifolia Lectin(BSL-1)</td>
			<td>Vector Labs</td>
			<td>L-1100</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG+IgM(H+L)</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-005-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgM (&#956;-chain)</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-005-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin-binding epidermal growth factor</td>
			<td>Sigma</td>
			<td>E4643</td>
			<td></td>
		</tr>
		<tr>
			<td>Human HepaCAM antibody</td>
			<td>R&#38;D systems</td>
			<td>MAB4108</td>
			<td></td>
		</tr>
		<tr>
			<td>Integrin beta 5 monoclonal antibody (KN52)</td>
			<td>eBioscience</td>
			<td>14-0497-82</td>
			<td></td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>Sigma</td>
			<td>C7880</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamate</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetly-L-cyteine (NAC)</td>
			<td>Sigma</td>
			<td>A8199</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal media</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>O4 hybridoma supernatant(mouse IgM)</td>
			<td>Bansal et al.23</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Worthington</td>
			<td>Is003126</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluristrainer 20 &#956;m</td>
			<td>PluriSelect</td>
			<td>43-50020-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>Sigma</td>
			<td>P6407</td>
			<td></td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P8783</td>
			<td></td>
		</tr>
		<tr>
			<td>Putrescine dihydrochloride</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified rat anti-mouse CD45</td>
			<td>BD Pharmingen</td>
			<td>550539</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified mouse anti-rat CD45</td>
			<td>BD Pharmingen</td>
			<td>554875</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium selenite</td>
			<td>Sigma</td>
			<td>S5261</td>
			<td></td>
		</tr>
		<tr>
			<td>Transferrin</td>
			<td>Sigma</td>
			<td>T1147</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma</td>
			<td>T9935</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin inhibitor</td>
			<td>Worthington</td>
			<td>LS003086</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-clear tube (Tube, Thinwall, Ultra-Clear)</td>
			<td>Beckman Coulter</td>
			<td>344059</td>
			<td></td>
		</tr>
		<tr>
			<td>Collect IP-ACM</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Macrosep Advance Centrifugal Devices with Omega Membrane (10k)</td>
			<td>PALL</td>
			<td>MAP010C37</td>
			<td></td>
		</tr>
		<tr>
			<td>Macrosep Advance Centrifugal Devices with Omega Membrane (30k)</td>
			<td>PALL</td>
			<td>MAP030C37</td>
			<td></td>
		</tr>
		<tr>
			<td>Phagocytosis live imaging assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Juli stage</td>
			<td>NanoEntek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Time Series Analyzer V3 plugins</td>
			<td>https://imagej.nih.gov/ij/plugins/time-series.html</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a novel in vitro live-imaging assay of astrocyte-mediated phagocytosis using ph indicator-conjugated synaptosomes

アストロ サイトは脳の主要な細胞型であり、シナプスや血管に直接お問い合わせください。アストロ サイトはまた発達シナプスの除去とのクリアランスなど、様々 な貪食プロセスに参加を最近の研究で示されているミクログリア細胞は、主要な免疫細胞と脳の食のみと考えられてきたがアルツハイマー病 (AD) におけるアミロイド β 斑。これらの調査結果にもかかわらずアストロ貪食の効率とそのターゲットの劣化は不明ミクログリアのそれと比較します。情報の欠如は、たいがいは、アストロ サイト、ミクログリアによる貪食能の動態が簡単に匹敵するアッセイ システムの欠如です。この目標を達成するためには、精製されたアストロ サイトやミクログリアの貪食能を評価する長期的なライブ イメージングの in vitro貪食能試の金を開発しました。このアッセイで貪食・分解のリアルタイム検出は、リソソームなどの酸性オルガネラの明るい赤の蛍光を発する pH インジケーター共役シナプトソームを使用して可能です。当社の新規アッセイは、ライブ イメージングによる貪食のシンプルで効果的な検出を提供します。さらに、このin vitro貪食アッセイは、化学物質とを強化したり、アストロ サイトの貪食能を阻害する化合物を識別するためにスクリーニングのプラットフォームとして使用できます。シナプスの刈り込みの異常と病原性蛋白質の蓄積は、精神疾患や神経変性疾患の原因と示されている、化学薬品およびグリア細胞の貪食能を調節する化合物を様々 な治療に役立つはず神経疾患。

このin vitro貪食のアッセイで長期的なライブ イメージングによる成体マウス脳ホモジネート遠心 (23% 勾配液と 10% 勾配ソリューションのグラデーションのソリューションで分離されたから得たシナプトソームを使いました図 3)。調製後シナプトソームは彼らが機能を失ったし、アストロ サイトやミクログリアにおける PS 受容体によって認...

Watch this Scientific Journal Video about A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes at JoVE.com

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

このプロトコルは、アストロ サイトの貪食能を測定するライブ イメージングの in vitro貪食アッセイを提示します。精製したラット アストロ サイトやミクログリアは、pH インジケーター共役シナプトソームと共に使用されます。このメソッドは、リアルタイムの貪食・分解速度を検出することができ、アストロ サイト細胞貪食の変調の要因を識別するために適切なスクリーニングのプラットフォームを提供します。

貪食能アッセイのアストロ サイトを介した新規体外ライブ イメージングを使用して pH インジケーター共役シナプトソーム

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major ...

a-novel-vitro-live-imaging-assay-astrocyte-mediated-phagocytosis

Research

JoVE Journal

Neuroscience

A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

12.0K ビュー.  Korea Advanced Institute of Science and Technology. このプロトコルは、アストロ サイトの貪食能を測定するライブ イメージングの in vitro貪食アッセイを提示します。精製したラット アストロ サイトやミクログリアは、pH インジケーター共役シナプトソームと共に使用されます。このメソッドは、リアルタイムの貪食・分解速度を検出することができ、アストロ サイト細胞貪食の変調の要因を識別...

ビデオ: A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (&alpha;, &beta;I, &beta;II and &gamma;), novel type I PKCs (&#949; and &eta;), novel type II PKCs (&delta; and &theta;), and atypical PKCs (&zeta; and &iota;). The classical PKCs are activated by Ca2+ and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca2+-independent. The atypical PKCs are activated by neither Ca2+ nor DAG. In Aplysia californica, our model system to study memory formation, there are three nervous system specific PKC isoforms one from each major class, namely the conventional PKC Apl I, the novel type I PKC Apl II and the atypical PKC Apl III. PKCs are lipid-activated kinases and thus activation of classical and novel PKCs in response to extracellular signals has been frequently correlated with PKC translocation from the cytoplasm to the plasma membrane. Therefore, visualizing PKC translocation in real time in live cells has become an invaluable tool for elucidating the signal transduction pathways that lead to PKC activation. For instance, this technique has allowed for us to establish that different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity and that serotonin (5HT) activation of PKC Apl II requires production of both DAG and phosphatidic acid (PA) for translocation 1-2. Importantly, the ability to visualize the same neuron repeatedly has allowed us, for example, to measure desensitization of the PKC response in exquisite detail 3. In this video, we demonstrate each step of preparing Sf9 cell cultures, cultures of Aplysia sensory neurons have been described in another video article 4, expressing fluorescently tagged PKCs in Sf9 cells and in Aplysia sensory neurons and live-imaging of PKC translocation in response to different activators using laser-scanning microscopy.

In this video, we demonstrate visualization of PKC translocation in living cells using fluorescently tagged PKCs.

Sf9細胞でとアメフラシ感覚ニューロンにおけるPKC転座のライブイメージング

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

神経科学

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

開発ゼブラフィッシュ前脳におけるクローン関連の神経前駆細胞のタイムラプスライブイメージング

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

根切断術後の後根の軸索のライブイメージング

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

ex vivoでマウス神経上皮における単一細胞分裂のライブイメージング

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

のライブイメージング<em&gt;ショウジョウバエ</em&gt;幼虫の神経芽細胞

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

マウスの脳の側脳室における上衣繊毛のライブイメージング

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

Novel Assay for Cold Nociception in Drosophila Larvae

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory machinery, such as in patients experiencing peripheral neuropathy or injury-induced sensitization, are not well characterized. The genetically tractable Drosophila model has been used to study the cells and genes required for noxious heat detection, which has yielded multiple conserved genes of interest. Little is known however about the cells and receptors important for noxious cold sensing. Although, Drosophila does not survive prolonged exposure to cold temperatures (&#8804;10 &#186;C), and will avoid cool, preferring warmer temperatures in behavioral preference assays, how they sense and possibly avoid noxious cold stimuli has only recently been investigated.
Here we describe and characterize the first noxious cold (&#8804;10 &#186;C) behavioral assay in Drosophila. Using this tool and assay, we show an investigator how to qualitatively and quantitatively assess cold nociceptive behaviors. This can be done under normal/healthy culture conditions, or presumably in the context of disease, injury or sensitization. Further, this assay can be applied to larvae selected for desired genotypes, which might impact thermosensation, pain, or nociceptive sensitization. Given that pain is a highly conserved process, using this assay to further study&#160;thermal nociception will likely glean important understanding of pain processes in other species, including vertebrates.

Novel Assay for Cold Nociception in Drosophila Larvae

Here we demonstrate a novel assay to study cold nociception in Drosophila larvae. This assay utilizes a custom-built Peltier probe capable of applying a focal noxious cold stimulus and results in quantifiable cold-specific behaviors. This technique will allow further cellular and molecular dissection of cold nociception.

冷痛覚のための新規アッセイ<em&gt;ショウジョウバエ</em&gt;幼虫

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

2次元培養系を用いた主な大脳皮質細胞のイメージングをライブします。

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

Bone marrow-derived macrophages (BMDMs) are mature leukocytes that serve a critical physiological role as professional phagocytes capable of clearing a variety of particles. Normally, BMDMs are restricted from the central nervous system (CNS), but following an injury, they can readily infiltrate. Once within the injured CNS tissue, BMDMs are the primary cell type responsible for the clearance of injury-derived cellular debris, including large quantities of lipid rich myelin debris. The neuropathological ramifications of BMDM infiltration and myelin debris phagocytosis within the CNS are complex and not well understood. The protocols described here, allow for the direct in vitro study of BMDMs in the context of CNS injury. We cover murine BMDM isolation and culture, myelin debris preparation, and assays to assess BMDM myelin debris phagocytosis. These techniques produce robust quantifiable results without the need for significant specialized equipment or materials, yet can be easily customized to meet the needs of researchers.

In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

We present methods to assess the phagocytic capacity of primary murine bone marrow-derived macrophages using fluorescently labeled myelin debris and intracellular lipid droplet staining.

体外骨髄由来マクロファージによって髄鞘の残骸の貪食

Bone marrow-derived macrophages (BMDMs) are mature leukocytes that serve a critical physiological role as professional phagocytes capable of clearing a ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

Sod1 突然変異体蛋白質集計における細胞の高いコンテンツ定量化のための分析法の開発

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

貪食能アッセイのアストロ サイトを介した新規体外ライブ イメージングを使用して pH インジケーター共役シナプトソーム

貪食能アッセイのアストロサイトを介した新規体外ライブイメージングを使用して pH インジケーター共役シナプトソーム