Name: a novel in vitro live-imaging assay of astrocyte-mediated phagocytosis using ph indicator-conjugated synaptosomes
Uploaded: 2018-02-05T13:30:00.000+00:00
Duration: 6 min 43 s
Description: 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

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>&lt;strong&gt;Synaptosome purification&lt;/strong&gt;</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>&lt;strong&gt;pH indicator conjugation&lt;/strong&gt;</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>&lt;strong&gt;Immunopanning&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10X Earle&amp;rsquo;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 (&amp;mu;-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&amp;amp;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.&lt;sup class=&quot;xref&quot;&gt;23&lt;/sup&gt;</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 &amp;mu;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>&lt;strong&gt;Collect IP-ACM&lt;/strong&gt;</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>&lt;strong&gt;Phagocytosis live imaging assay&lt;/strong&gt;</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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A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

12.1K 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

An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and subsequently digests this material, commonly through lysosomal degradation. Microglia are the resident immune cells of the central nervous system (CNS) whose phagocytic function has been described in a broad range of conditions from neurodegenerative disease (e.g., beta-amyloid clearance in Alzheimer&#8217;s disease) to development of the healthy brain (e.g., synaptic pruning)1-6. The following protocol is an engulfment assay developed to visualize and quantify microglia-mediated engulfment of presynaptic inputs in the developing mouse retinogeniculate system7. While this assay was used to assess microglia function in this particular context, a similar approach may be used to assess other phagocytes throughout the brain (e.g., astrocytes) and the rest of the body (e.g., peripheral macrophages) as well as other contexts in which synaptic remodeling occurs (e.g. ,brain injury/disease).

Microglia are the resident immune cells of the central nervous system (CNS) with a high capacity to phagocytose or engulf material in their extracellular environment. Here, a broadly applicable, reliable, and highly quantitative assay for visualizing and measuring microglia-mediated engulfment of synaptic components is described.

말림 분석 : CNS 식세포와 신경 세포 사이의 상호 작용을 평가하기위한 프로토콜

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and ...

연구

신경과학

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

3 차원 조직 설계 정렬 사이토 네트워크 발달 메커니즘을 정리 하 고 신경 재생을 촉진 하

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

생체공학

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

망막 미세 아교 식세포의 기능을 평가<em&gt; 생체</em&gt;은 유동 세포 계측법 기반 분석을 사용하여

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

면역학 및 감염병학

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of oxygen and glucose, which causes energy failure and neuronal death. After an ischemic stroke insult, astrocytes become reactive and proliferate around the injury site as it develops. Under this scenario, it is difficult to study the specific contribution of astrocytes to the brain region exposed to ischemia. Therefore, this article introduces a methodology to study primary astrocyte reactivity and proliferation under an in vitro model of an ischemia-like environment, called oxygen glucose deprivation (OGD). Astrocytes were isolated from 1-4 day-old neonatal rats and the number of non-specific astrocytic cells was assessed using astrocyte selective marker Glial Fibrillary Acidic Protein (GFAP) and nuclear staining. The period in which astrocytes are subjected to the OGD condition can be customized, as well as the percentage of oxygen they are exposed to. This flexibility allows scientists to characterize the duration of the ischemic-like condition in different groups of cells in vitro. This article discusses the timeframes of OGD that induce astrocyte reactivity, hypertrophic morphology, and proliferation as measured by immunofluorescence using Proliferating Cell Nuclear Antigen (PCNA). Besides proliferation, astrocytes undergo energy and oxidative stress, and respond to OGD by releasing soluble factors into the cell medium. This medium can be collected and used to analyze the effects of molecules released by astrocytes in primary neuronal cultures without cell-to-cell interaction. In summary, this primary cell culture model can be efficiently used to understand the role of isolated astrocytes upon injury.

Monitoring Astrocyte Reactivity and Proliferation in Vitro Under Ischemic-Like Conditions

Ischemic stroke is a complex event in which the specific contribution of astrocytes to the affected brain region exposed to oxygen glucose deprivation (OGD) is difficult to study. This article introduces a methodology to obtain isolated astrocytes and study their reactivity and proliferation under OGD conditions.

사이토 반응성 및 확산에서 체 외에 허 혈 성 같은 조건 모니터링

Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of ...

Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in brain homeostasis, astrocytes supply neurons with vital metabolites and buffer extracellular water, ions, and glutamate. An integral component of the &#8220;tri-partite&#8221; synapse, astrocytes are also critical in the formation, pruning, maintenance, and modulation of synapses. To enable these highly interactive functions, astrocytes communicate among themselves and with other glial cells, neurons, the brain vasculature, and the extracellular environment through a multitude of specialized membrane proteins that include cell adhesion molecules, aquaporins, ion channels, neurotransmitter transporters, and gap junction molecules. To support this dynamic flux, astrocytes, like neurons, rely on tightly coordinated and efficient intracellular transport. Unlike neurons,&#160;where intracellular trafficking has been extensively delineated, microtubule-based transport in astrocytes has been less studied. Nonetheless, exo- and endocytic trafficking of cell membrane proteins and intracellular organelle transport orchestrates astrocytes&#8217; normal biology, and these processes are often affected in disease or in response to injury. Here we present a straightforward protocol to culture high quality murine astrocytes, to fluorescently label astrocytic proteins and organelles of interest, and to record their intracellular transport dynamics using time-lapse confocal microscopy. We also demonstrate how to extract and quantify relevant transport parameters from the acquired movies using available image analysis software (i.e., ImageJ/FIJI) plugins.

Here we describe an in vitro live-imaging method to visualize intracellular transport of organelles and trafficking of plasma membrane proteins in murine astrocytes. This protocol also presents an image analysis methodology to determine cargo transport itineraries and kinetics.

성상 세포에서 세포기관 및 기타 화물의 세포내 운송 시각화 및 분석

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in ...

In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. Microglia clear up dead and dying neurons through the process of efferocytosis, a specialized form of phagocytosis. The phagocytosis function can be disrupted by environmental or genetic risk factors that affect microglia. This paper presents a rapid and simple in vitro&#160;microscopy protocol for studying microglial efferocytosis in an induced pluripotent stem cell (iPSC) model of microglia, using a human neuroblastoma cell line (SH-SY5Y) labeled with a pH-sensitive dye for the phagocytic cargo. The procedure results in a high yield of dead neuroblastoma cells, which display surface phosphatidylserine, recognized as an &#34;eat-me&#34; signal by phagocytes. The 96-well plate assay is suitable for live-cell time-lapse imaging, or the plate can be successfully fixed prior to further processing and quantified by high-content microscopy. Fixed-cell high-content microscopy enables the assay to be scaled up for screening of small molecule inhibitors or assessing the phagocytic function of genetic variant iPSC lines. While this assay was developed to study phagocytosis of whole dead neuroblastoma cells by iPSC-macrophages, the assay can be easily adapted for other cargoes relevant to neurodegenerative diseases, such as synaptosomes and myelin, and other phagocytic cell types.

Neurodegenerative diseases are associated with dysregulated microglia functions. This article outlines an in vitro assay of phagocytosis of neuroblastoma cells by iPSC-macrophages. Quantitative microscopy readouts are described for both live-cell time-lapse imaging and fixed-cell high-content imaging.

iPSC-대식세포에 의한 죽은 신경 모세포종 세포의 식증에 대한 체외 정량적 이미징 분석

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. ...

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple neurological diseases. Studying human microglia in health and disease represents a challenge due to the extremely limited supply of human cells. Induced pluripotent stem cells (iPSCs) derived from human individuals can be used to circumvent this barrier. Here, it is demonstrated how to differentiate human iPSCs into microglia-like cells (iMGs) for in vitro experimentation. These iMGs exhibit the expected and physiological properties of microglia, including microglia-like morphology, expression of proper markers, and active phagocytosis. Additionally, documentation for isolating and labeling synaptosome substrates derived from human iPSC-derived lower motor neurons (i3LMNs) is provided. A live-cell, longitudinal imaging assay is used to monitor engulfment of human synaptosomes labeled with a pH-sensitive dye, allowing for investigations of iMG's phagocytic capacity. The protocols described herein are broadly applicable to different fields that are investigating human microglia biology and the contribution of microglia to disease.

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

This protocol describes the differentiation process of human induced pluripotent stem cells (iPSCs) into microglia-like cells for in vitro experimentation. We also include a detailed procedure for generating human synaptosomes from iPSC-derived lower motor neurons that can be used as a substrate for in vitro phagocytosis assays using live-cell imaging systems.

인간 미세아교세포 유사 세포: 유도만능줄기세포로부터의 분화 및 인간 시냅토좀을 이용한 체외 생세포 식균작용 분석

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...

시냅스 물질의 미세아교세포 삼킴을 위한 유세포분석 기반 분석

Quantification of Microglial Engulfment of Synaptic Material Using Flow Cytometry

Microglia play a pivotal role in synaptic refinement in the brain. Analysis of microglial engulfment of synapses is essential for comprehending this process; however, currently available methods for identifying microglial engulfment of synapses, such as immunohistochemistry (IHC) and imaging, are laborious and time-intensive. To address this challenge, herein we present in vitro and in vivo* assays that allow fast and high-throughput quantification of microglial engulfment of synapses using flow cytometry.
In the in vivo* approach, we performed intracellular vGLUT1 staining following fresh cell isolation from adult mouse brains to quantify engulfment of vGLUT1+ synapses by microglia. In the in vitro synaptosome engulfment assay, we used freshly isolated cells from the adult mouse brain to quantify the engulfment of pHrodo&#160;Red-labeled synaptosomes by microglia. These protocols together provide a time-efficient approach to quantifying microglial engulfment of synapses and represent promising alternatives to labor-intensive image analysis-based methods. By streamlining the analysis, these assays can contribute to a better understanding of the role of microglia in synaptic refinement in different disease models.

저자 스포트라이트: 정신신경면역학의 기술 혁신을 통한 이해 증진

Here we present two protocols to quantify microglial engulfment of vGLUT1-positive synapses and pHRodo&#160;Red-labeled crude synaptosomes using flow cytometry.

유세포분석을 사용한 시냅스 물질의 미세아교세포 삼킴 정량화

Microglia play a pivotal role in synaptic refinement in the brain. Analysis of microglial engulfment of synapses is essential for comprehending this ...

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

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말림 분석 : CNS 식세포와 신경 세포 사이의 상호 작용을 평가하기위한 프로토콜

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망막 미세 아교 식세포의 기능을 평가

사이토 반응성 및 확산에서 체 외에 허 혈 성 같은 조건 모니터링

성상 세포에서 세포기관 및 기타 화물의 세포내 운송 시각화 및 분석

iPSC-대식세포에 의한 죽은 신경 모세포종 세포의 식증에 대한 체외 정량적 이미징 분석

인간 미세아교세포 유사 세포: 유도만능줄기세포로부터의 분화 및 인간 시냅토좀을 이용한 체외 생세포 식균작용 분석

유세포분석을 사용한 시냅스 물질의 미세아교세포 삼킴 정량화

유세포분석을 사용한 시냅스 물질의 미세아교세포 삼킴 정량화

유세포분석을 사용한 시냅스 물질의 미세아교세포 삼킴 정량화