저자 감사 연-주 정 그녀의 실험적인 지원에 대 한 synaptosome 정화와 Jungjoo 공원 동안 PS 노출 synaptosomes의 이미지. 또한, 우리는 유용한 토론에 대 한 정의 실험실에서 모든 회원을 감사합니다. 이 작품은 한국 정부 (MSIP) (NRF 2016M3C7A1905391와 NRF-2016R1C1B3006969)에 의해 투자 하는 한국 (NRF) 부여의 국립 연구 재단에 의해 지원 되었다 (W. S. C)입니다.

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저자는 공개 없다.

이 문서에서 우리는 장기 생체 외에서 라이브 이미징 먹어서 분석 결과 정제 glial 세포 및 pH 지시자 활용 된 synaptosomes를 사용 하 여 위한 방법을 제시. 우리 microglia와 비교 하는 표시 이다 synaptosomes의 식 균 작용 동안 다른 engulfment 저하 용량을가지고. 또한, 우리의 데이터 사이토 분 비 요인, GAS6, 단백질, 및 MEGE8 같은 브리징 분자를 포함 하는 두뇌에 있는 glial 세포의 효율적인 PS 종속 먹어서...

두뇌에 고르기가 아닌 셀 참조, glial 세포 중앙 신경 시스템 (CNS)에 주요 셀 유형입니다. 이전, glial 세포는 시 냅 스 속성과 기저 신경 생존 유지에 수동 역할 주로 단순한 지원 세포로 간주 되었다. 그러나, glial 세포 신경 생물학, 뇌 항상성, 중재 시 냅 스 형성1,2,3 , 시 냅 스를 유지 하는 등의 다양 한 측면에서 보다 적극적인 역할을 재생할 계시는 증거를 신흥 제거4,5, 그리고 변조 시 냅 스가 소성6,7. CNS에서 glial 세포는 이다, microglia, 및 oligodendrocytes 포함 됩니다. 이러한 세포, 이다와 microglia 보였다 시 냅 스4,5, apoptotic 세포8, 신경 파편9, 그리고 병원 성 단백질, 녹말 체 beta 등을 덮어 여 phagocytic 역할을 플 라크10,11. 개발 두뇌에서 이다 MERTK 및 MEGF10 종속 먹어서4등 측면 geniculate 핵 (dLGN)에서 시 냅 스를 제거합니다. 마찬가지로, microglia 또한 C1q 코팅 시 냅 스 클래식 보완 캐스케이드5발달 단계를 제거 합니다. 흥미롭게도, 그것은 결함 시 냅 스의 가지 치기에 여러 가지 신경 장애의 시작자 중 하나 될 수 있습니다 제안 되었습니다. 예를 들어 돌연변이 보완 구성 요소 4에서에서 (C4) microglia에 의해 보완-중재 시 냅 스의 가지 치기를 증가 인간12정신 분열 증의 보급에 강하게 연관 표시 되었습니다. 최근 종이 고아 한 보충 통로 hyperactivated 광고의 개시 단계에 하 고13이 질병에서 초기 냅 손실 유도도 표시 됩니다.
먹어서 microglia 중재와 비교, 덜 분명 하다 사이토 중재 먹어서 개시와 다양 한 신경 질환의 진행에 기여 하는 여부. 그러나, 최근 종이 이다 하 여 정상적인 시 냅 스 가지 치기의 속도 변경 하는 수 있습니다 두뇌 항상성 방해 요인과 광고 민감성과 병리학14을 나왔다. ApoE 이 성체, 광고 (ApoE2)에 대 한 보호 대립 유전자와 강하게 향상 속도 위험 대립 유전자 (ApoE4) 광고에 대 한 속도 크게 낮추는 이다에 의해 시 냅 스 가지 치기의 속도 강력 하 게 제어 됩니다. 또한, 유전자 변형 마우스 ApoE4 표현 제어 또는 ApoE2 마우스14보다 훨씬 더 많은 시 냅 스 C1q 축적. 이러한 데이터는 초기에 사이토 중재 먹어서 장애인 제안 광고 두뇌 활성화 보완-중재 microglial 먹어서, 시 냅 스 변성 운전 노화 C1q 코팅 시 냅 스/시 냅 스 파편의 축적을 유발 수 있습니다 . ApoE4 운반대에 이다의 장애인된 phagocytic 용량 또한 광고 영향을 두뇌에 베타 아 밀 로이드 플 라크의 통제 축적에 기여할 수 있습니다.
또한, 그것은 세 초파리 뇌에 glial 세포 드레이퍼, Megf10 이다 phagocytosing 시 냅 스를 사용 하는의 체의 감소 번역으로 인해 그들의 phagocytic 용량을 잃게 표시 되었습니다. 효율적으로 비슷한 정도 젊은 뇌를 노화 유도 나타내는 세 뇌에 손상된 axonal 파편 삭제 glial 세포의 phagocytic 용량 구조 드레이퍼 수준을 회복의 phagocytic 용량 변경 이다 두뇌 항상성15의 파괴에 기여할 수 있습니다.
이러한 새로운 발견을 바탕으로, 이다의 phagocytic 용량 변조 수 방지 하 고 다양 한 신경 질환을 치료 하는 매력적인 치료 전략. 이와 관련, 산 성 나노16 리소좀의 산성화를 유발 및 녹음 방송 요인 EB (TFEB), 향상 시킬 수 있는 overexpressing 이다, 예를 들어의 phagocytic 능력을 향상 시키는 몇 가지 시도 되었습니다. 리소좀 속17. 이러한 시도도 불구 하 고 그것은 여전히 확실치있지 않습니다 어떻게 이다 및 microglial 셀 phagocytic 그들의 활동에 다 우리가 해야 증가 여부 등 다양 한 질병에 그들의 phagocytic 용량 감소.
이 논문에서는, 소설 체 외에서 분석 결과 실시간으로 이다의 phagocytic 용량을 탐지 하기 위한 선물이. 데이터 이다에 microglia engulfment과 저하의 다른 활동을 보여줍니다. 사이토 조절 매체 (ACM), 포함 이다에서 분 비 요인, 이다와 microglia의 효과적인 먹어서 필수적입니다. 또한, Megf10, 이다에 Ced-1 과 드레이퍼, 체 phagocytic 수용 체 중재 하는 사이토 먹어서8,18에 중요 한 역할을 재생 합니다.

<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

이다 두뇌에 있는 주요한 세포 유형 고 시 냅 스 및 혈관에 직접 문의. Microglial 세포는 주요 면역 세포와 뇌의 식 세포만 간주 되었습니다, 하지만 최근의 연구 이다 또한 개발 시 냅 스 제거 등의 다양 한 phagocytic 프로세스에 참여 Alzheimer의 질병 (광고)에서 베타 아 밀 로이드 플 라크. 이러한 결과도 불구 하 고 사이토 engulfment의 효율성 및 그들의 목표의 명확 하지 않다 microglia의와 비교. 정보의이 부족 있는 사이토 microglia 중재 먹어서의 활동은 쉽게 비교 분석 결과 시스템의 부족 때문에 주로 이다. 이 목표를 달성 하기 위해 장기 생체 외에서 라이브 이미징 먹어서 분석 결과 순화 이다와 microglia phagocytic 용량 평가를 개발 했습니다. 이 분석 결과에 engulfment과 저하의 실시간 탐지 표시기 활용 된 synaptosomes, 리소좀과 같은 산 성 세포에 밝은 적색 형광을 방출 하는 pH를 사용 하 여 가능 하다. 우리의 새로운 분석 결과 라이브 영상 통해 식 균 작용의 간단 하 고 효과적인 검색을 제공합니다. 또한, 화학 물질 및 향상 시킬 수 또는 이다의 phagocytic 용량을 억제 하는 화합물을 식별 하이 체 외에서 먹어서 분석 결과 심사 플랫폼으로 사용할 수 있습니다. 화학 물질 및 화합물 glial 세포의 phagocytic 용량을 조절 하는 다양 한 치료에 도움이 될 것으로 시 냅 스 가지 치기 오작동 및 병원 성 단백질 축적 정신 장애 또는 신경 퇴행 성 질환 원인 표시 되었습니다. 신경 장애입니다.

이 생체 외에서 먹어서 분석 결과 장기 라이브 이미징,에서는 사용 하는 ultracentrifugation (23% 그라데이션 솔루션 및 10% 그라데이션 솔루션 사이의 그라데이션 솔루션에서 분리 했다 성인 마우스 뇌 homogenates에서 synaptosomes 그림 3)입니다. 준비 후에, synaptosomes는 그들은 그들의 기능을 잃은 것 이다 및 microglia PS 수용 체에 의해 인식 될 수 있는 제안...

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

이 프로토콜 생체 외에서 라이브 이미징 먹어서 분석 결과 이다 phagocytic 용량의 측정을 제공 합니다. Microglia 및 순화 된 쥐 이다 pH 지시자 활용 된 synaptosomes 함께 사용 됩니다. 이 방법은 실시간 engulfment과 저하 속도 검색할 수 있습니다 하 고 사이토 식 균 작용을 조절 하는 요소를 식별 하는 적합 한 심사 플랫폼을 제공 합니다.

소설 체 외에 라이브 이미징 분석 결과의 사이토 중재 먹어서 사용 하 여 pH 지시자 활용 Synaptosomes

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

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Neuroscience

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

12.0K 조회수.  Korea Advanced Institute of Science and Technology. 이 프로토콜 생체 외에서 라이브 이미징 먹어서 분석 결과 이다 phagocytic 용량의 측정을 제공 합니다. Microglia 및 순화 된 쥐 이다 pH 지시자 활용 된 synaptosomes 함께 사용 됩니다. 이 방법은 실시간 engulfment과 저하 속도 검색할 수 있습니다 하 고 사이토 식 균 작용을 조절 하는 요소를 식별 하는 적합 한 심사 플랫폼을 제공 합니다.

비디오: 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 세포에서 Aplysia 감각 뉴런에서 PKC Translocation의 라이브 영상

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 ...

연구

신경과학

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.

개발 Zebrafish Forebrain에 Clonally 관련 신경 전구 세포의 시간 경과 라이브 영상

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.

Rhizotomy 후 도설 루트 Axons의 라이브 영상

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

생체 마우스 Neuroepithelium에서 단일 세포 분열의 라이브 영상

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.

의 라이브 영상<em&gt; 초파리</em&gt; 애벌레 Neuroblasts

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.

마우스 뇌의 좌우 심실의 뇌실막 섬모의 라이브 영상

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.

콜드 통각에 대한 소설 분석<em&gt; 초파리</em&gt; 애벌레

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.

2D 문화 시스템을 사용 하 여 기본 대뇌 피 질 세포의 영상 라이브

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.

생체 외에서 골 수 유래 세포에 의해 Myelin 파편 식 균 작용

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.

살아있는 세포에 Sod1 돌연변이 단백질 집계 형성의 높은 콘텐츠 정량화에 대 한 분석 결과 개발

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

소설 체 외에 라이브 이미징 분석 결과의 사이토 중재 먹어서 사용 하 여 pH 지시자 활용 Synaptosomes

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