Yazarlar Yeon-Joo Jung onun deneysel destek için synaptosome arınma ve Jungjoo Park sırasında synaptosomes PS pozlama ile yansımaları için teşekkür ederiz. Buna ek olarak, tüm üyelerinde Chung'ın laboratuvar yararlı tartışma için teşekkür ederiz. Bu eser Kore (NMG) hibe Kore hükümeti (MSIP) (NMK-2016M3C7A1905391 ve NATO Mukabele Gücü-2016R1C1B3006969) tarafından finanse edilen Ulusal Araştırma Vakfı tarafından desteklenmiştir (W. S. C).

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Yazarlar ifşa gerek yok.

Bu makalede, biz yöntemleri arıtılmış gliyal hücreler ve pH göstergesi Birleşik synaptosomes kullanarak uzun vadeli bir canlı görüntüleme vitro fagositoz tahlil için mevcut. Biz microglia ile karşılaştırıldığında göstermek astrocytes synaptosomes fagositoz sırasında farklı engulfment ve yıkımı kapasitesine sahip. Buna ek olarak, bizim veri köprüleme moleküllerin GAS6, proteinler ve MEGE8 içeren astrocyte salgıladığı faktörler, verimli PS-bağımlı fagositoz beyinde gliyal h?...

Beyinde telaşlı olmayan hücrelere başvuruda bulunan, gliyal hücreler merkezi sinir sistemi (MSS) büyük hücre tipinde vardır. Daha önce gliyal hücreler esas olarak nöronal hayatta kalma ve Bazal sinaptik özellikleri korumak pasif rol oynarlar sadece destekleyen hücreler olarak kabul edildi. Ancak, kanıt ortaya çıkan gliyal hücreler Nörobiyoloji synapse oluşumu1,2,3 ve synapse arabuluculuk beyin homeostazı korumak gibi çeşitli yönleriyle daha aktif rol oynarlar ortaya koymuştur Eleme4,5ve modülasyonlu sinaptik plastisite6,7. MSS gliyal hücreler astrocytes, microglia ve oligodendrocytes içerir. Bu hücreler, astrocytes ve microglia arasında sinapslarda4,5, apoptotik hücreler8, sinirsel enkaz9ve patojenik proteinler, amiloid beta gibi içine çeken fagositik oynamamızgerektiğini gösterilmiştir plaklar10,11. Gelişmekte olan beyin astrocytes dorsal yanal geniculate çekirdek (dLGN) ile MERTK ve MEGF10 bağımlı fagositoz4sinapslarda ortadan kaldırmak. Benzer şekilde, microglia da C1q kaplı sinapslarda ile klasik Kompleman art arda5gelişim dönemleri sırasında ortadan. İlginçtir, ekranlarda synapse budama bulunan çeşitli nörolojik bozukluklar başlatanlar biri olabilir sürülmüştür. Örneğin, tamamlayıcı bileşenindeki 4 mutasyonlar (C4), Kompleman aracılı synapse budama microglia tarafından artırır, güçlü insanlar12şizofreni yaygınlığı ile ilişkili olan gösterilmiştir. Son bir kağıt da klasik Kompleman yolu hyperactivated reklam başlatma aşamasında olduğunu ve bu hastalığı13erken synapse kaybına neden olmaktadır göstermiştir.
Microglia-aracılı fagositoz ile karşılaştırıldığında, astrocyte-aracılı fagositoz başlama ve ilerleme çeşitli nörolojik bozukluk olup katkıda bulunur o kadar net değildir. Ancak, son bir kağıt astrocytes tarafından normal synapse budama hızını değiştiren faktörler beyin homeostazı bozabilir ve reklam duyarlılık ve patoloji14' e katkıda öneriyor. Synapse budama astrocytes tarafından oranı güçlü güçlü artırılması oranı ve reklam (ApoE4) için bir risk alleli hızını önemli ölçüde azaltmayı ApoE İzomerler tarafından reklam (ApoE2) için koruyucu bir gen ile kontrol edilir. Ayrıca, ApoE4 ifade transgenik fareler denetim veya ApoE2 fareler14daha fazla sinaptik C1q birikmiş. Bu astrocyte-aracılı fagositoz erken engelliler bu verileri göstermektedir reklam beyin mikroglial Kompleman aracılı fagositoz, sinaptik bozulma sürüş etkinleştirir senescent C1q kaplı sinapslarda/sinaptik enkaz birikimi neden . Astrocytes ApoE4 taşıyıcıları olarak Engelli fagositik kapasite Ayrıca reklam etkilenen beynindeki beta amiloid plaklar kontrolsüz birikimi için katkıda bulunabilir.
Buna ek olarak, gliyal yaşlı Drosophila beyin hücrelerinde fagositik kapasitelerini Draper, sinapslarda phagocytosing için astrocytes kullanan Megf10 homolog azalmış çeviri nedeniyle kaybeder gösterilmiştir. Draper düzeyleri geri kurtarıldı verimli bir şekilde bu yaşlanma indüklenen gösteren benzer ölçüde bu kadar genç beyin, yaşlı beyindeki hasarlı aksonal enkaz temizlenmiş gliyal hücreler fagositik kapasite değişiklikler fagositik kapasite astrocytes beyin homeostazı15kesintileri için katkıda bulunabilir.
Bu yeni bulgulara dayanarak, astrocytes fagositik kapasite oransal önlemek ve çeşitli sinir hastalıkları tedavi için çekici bir tedavi stratejisi olabilir. Bu bağlamda, asidik nano tanecikleri16 ile organellerin asitleştirme inducing ve transkripsiyon faktörü EB (TFEB), hangi-ebilmek arttırmak overexpressing astrocytes, örneğin, fagositik kapasite geliştirmek için çeşitli girişimler olmuştur lysosome Biyogenez17. Bu girişimlerine rağmen nasıl astrocytes ve mikroglial hücreleri fagositik onların Kinetik farklı ve olup olmadığını biz azaltmak veya artırmak çeşitli hastalıklarında fagositik kapasitelerini hala belli değil.
Bu yazıda, biz astrocytes fagositik kapasite gerçek zamanlı olarak algılamak için bir roman vitro tahlil mevcut. Verileri farklı Kinetik engulfment ve yıkımı astrocytes ve microglia göster. Salgılanan faktörlerden yola çıkarak astrocytes içeren astrocyte şartına orta (ACM), astrocytes ve microglia etkili fagositoz için esastır. Ayrıca, Megf10, astrocytes ve homolog Ced-1 ve Draper, fagositik reseptör çalış önemli roller astrocyte-aracılı fagositoz8,18.

<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

Astrocytes beyin büyük hücre türü ve sinapslarda ve kan damarları doğrudan bağlantı kurun. Ne kadar büyük bağışıklık hücreleri ve sadece fagositler beyin mikroglial hücreleri olarak, son yıllarda yapılan çalışmalarda astrocytes da gelişimsel synapse eleme ve gümrükleme gibi çeşitli fagositik süreçlerde katılmak göstermiştir Alzheimer hastalığı (Ah) içinde beta amiloid plaklar. Bu bulgular rağmen astrocyte engulfment verimliliğini ve yıkımı hedeflerinin microglia ile karşılaştırıldığında belirsizdir. Çoğunlukla astrocyte ve microglia aracılı fagositoz Kinetik kolayca karşılaştırılabilir bir tahlil sistemi olmaması nedeniyle bilgi bu olmaması. Bu hedefe ulaşmak için saf astrocytes ve microglia fagositik kapasitesini değerlendirmek için uzun vadeli bir canlı görüntüleme vitro fagositoz tahlil geliştirdik. Bu tahlil, engulfment ve yıkımı gerçek zamanlı algılama gibi organellerin asidik organelleri parlak kırmızı Floresans çıkaran pH göstergesi Birleşik synaptosomes, kullanarak mümkündür. Bizim roman tahlil fagositoz canlı görüntüleme ile basit ve etkili olarak algılanmasını sağlar. Buna ek olarak, bu vitro fagositoz tahlil bir tarama platform olarak kimyasallar ve artırabilir veya astrocytes fagositik kapasite inhibe bileşikler tanımlamak için kullanılabilir. Sinaptik budama arıza ve patojenik protein birikimine ruhsal bozukluklar veya nörodejeneratif hastalıklar neden gösterilmiştir gibi kimyasallar ve gliyal hücreler fagositik kapasite modüle bileşikler çeşitli tedavisinde yararlı olmalıdır nörolojik bozukluklar.

Bu vitro fagositoz assay olarak uzun vadeli canlı görüntüleme, biz üzerinden % 23 degrade çözüm ve % 10 degrade çözüm arasında degrade çözümde ayrı kaldık ultrasantrifüj () tarafından Yetişkin fare beyin homogenates synaptosomes kullanılan Şekil 3). Hazırlık sonra telkin onların işlevini kaybetti ve PS reseptörleri astrocytes ve microglia tarafından tanınan PS onların dış membran (şekil 4...

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

Bu iletişim kuralı bir vitro canlı görüntüleme fagositoz tahlil astrocytes fagositik kapasitesini ölçmek için sunar. Arıtılmış sıçan astrocytes ve microglia pH göstergesi Birleşik synaptosomes ile birlikte kullanılır. Bu yöntem gerçek zamanlı engulfment ve yıkımı Kinetik algılayabilir ve astrocyte fagositoz oransal faktörleri tanımlamak için uygun tarama platformu sağlar.

Bir roman In Vitro canlı görüntüleme Astrocyte tahlil-aracılı fagositoz kullanarak pH göstergesi Birleşik 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 Görüntüleme.  Korea Advanced Institute of Science and Technology. Bu iletişim kuralı bir vitro canlı görüntüleme fagositoz tahlil astrocytes fagositik kapasitesini ölçmek için sunar. Arıtılmış sıçan astrocytes ve microglia pH göstergesi Birleşik synaptosomes ile birlikte kullanılır. Bu yöntem gerçek zamanlı engulfment ve yıkımı Kinetik algılayabilir ve astrocyte fagositoz oransal faktörleri tanımlamak için uygun tarama platformu sağlar.

Video: 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.

Bir yutulma Deneyi: CNS Fagositler ve nöronlar arasındaki etkileşimi değerlendirmek için bir protokol

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

Nörobilim

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.

Astrocyte ağlar gelişimsel mekanizmaları özetlemek ve sinir sistemi yeniden oluşturma işlemi kolaylaştırmak için tasarlanmış üç boyutlu doku uyumlu

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

Biyomühendislik

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.

Retina Mikroglial Fagositik Fonksiyonu değerlendirilmesi<em&gt; İn Vivo</em&gt; A Akış Sitometri bazlı analiz kullamlarak

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

İmmünoloji ve Enfeksiyon

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.

Astrocyte reaktivite ve nükleer silahların yayılmasına karşı Vitro iskemik benzeri koşullar altında izleme

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.

Astrositlerde Organeller ve Diğer Kargoların Hücre İçi Taşınmasının Görselleştirilmesi ve Analizi

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.

İPSC-Makrofajlar tarafından Ölü Nöroblastom Hücrelerinin Fagositozu için İn vitro Nicel Görüntüleme Tahlili

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.

İnsan Mikroglia Benzeri Hücreler: İnsan Sinaptozomları Kullanılarak İndüklenmiş Pluripotent Kök Hücrelerden ve In Vitro Canlı Hücre Fagositoz Analizinden Farklılaşma

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

Sinaptik Materyallerin Mikroglial Yutulması için Akış Sitometrisi Tabanlı Testler

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.

Yazar Spotlight: Psikonöroimmünolojide Teknolojik Yenilikler Yoluyla Anlayışı Geliştirme

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

Akış Sitometrisi Kullanılarak Sinaptik Materyalin Mikroglial Yutulmasının Miktar Tayini

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

Bir roman In Vitro canlı görüntüleme Astrocyte tahlil-aracılı fagositoz kullanarak pH göstergesi Birleşik Synaptosomes

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Bir yutulma Deneyi: CNS Fagositler ve nöronlar arasındaki etkileşimi değerlendirmek için bir protokol

Astrocyte ağlar gelişimsel mekanizmaları özetlemek ve sinir sistemi yeniden oluşturma işlemi kolaylaştırmak için tasarlanmış üç boyutlu doku uyumlu

Retina Mikroglial Fagositik Fonksiyonu değerlendirilmesi

Astrocyte reaktivite ve nükleer silahların yayılmasına karşı Vitro iskemik benzeri koşullar altında izleme

Astrositlerde Organeller ve Diğer Kargoların Hücre İçi Taşınmasının Görselleştirilmesi ve Analizi

İPSC-Makrofajlar tarafından Ölü Nöroblastom Hücrelerinin Fagositozu için İn vitro Nicel Görüntüleme Tahlili

İnsan Mikroglia Benzeri Hücreler: İnsan Sinaptozomları Kullanılarak İndüklenmiş Pluripotent Kök Hücrelerden ve In Vitro Canlı Hücre Fagositoz Analizinden Farklılaşma

Akış Sitometrisi Kullanılarak Sinaptik Materyalin Mikroglial Yutulmasının Miktar Tayini

Akış Sitometrisi Kullanılarak Sinaptik Materyalin Mikroglial Yutulmasının Miktar Tayini

Akış Sitometrisi Kullanılarak Sinaptik Materyalin Mikroglial Yutulmasının Miktar Tayini