Name: selective depletion of microglia from cerebellar granule cell cultures using l-leucine methyl ester
Uploaded: 2015-07-07T15:00:00.000+00:00
Duration: 8 min 10 s
Description: Watch this Scientific Journal Video about Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester at JoVE.com

This research was support by an Aims2Cure, UK and a UCL Impact Award Ph.D. studentship to JMP and an MRC Capacity Building Ph.D. studentship in Dementia to JMP.

本明細書に記載の全ての実験は、1986年の英国動物（科学的手続）法に基づいて実施しました。 楽器、文化メディア、および料理の作製<ol><li> 2つのステンレス鋼実験室解剖ハサミと2つのステンレス鋼実験用鉗子を準備します。紫外線下で培養フードO / Nですべての機器と場所をオートクレーブ滅菌用（UV）光。 </li><li>最小必須培地（MEM）培地（10％ウシ胎児血清[FBS]、20mMの塩化カリウム、25mMのNaHCO 3を、30mMのD-グルコース、2mMのL-グルタミン、100 U / mlペニシリン、100 500mlのを作ります/ mlのストレプトマイシンおよび6 / mlのアンピシリン）とフィルター滅菌します。ヶ月間4℃で保存してください。 </li><li> 14ミリメートル、数1の厚さのカバースリップを含む24ウェル培養プレートを準備します。先に説明したように3 100 mg / Lのポリ-D-リジン（PDL）でカバースリップおよびコーティングをオートクレーブ。 UV光はO / Nを殺菌します。 </li></ol> CGC培養液の調製<ol><li>処理されたオートクレーブ処理、UV光の「溶液A」（100ミリリットル）、グルコース250mgのを含有する無菌のdH 2 O、ウシ血清アルブミン[BSA]、リン酸緩衝生理食塩水[PBS]（のCa 2+の955 mgの300 mgを準備し、 MG）+自由、および3.8％の1ミリリットルのMgSO 4·7H 2 O </li><li> 5 mg / mlのトリプシン溶液Aの9ミリリットル中に希釈し、1 mlを含む準備「溶液B」（10ミリリットル） </li><li> 「溶液C」1000個のDNアーゼ、大豆トリプシン阻害剤0.5mgの、溶液Aで10mlに希釈した3.8％のMgSO 4·7H 2 Oの100μLを含む（10ml）に準備</li><li>溶液C 3.2 mlを含有する「溶液D &#39;（20ml）を溶液Aで20mlまで希釈し、準備します</li><li> ·7H 2 O EBSS、25mMのNaHCO 3を、3 mMの硫酸マグネシウムを含むBSA /アールの平衡塩溶液（EBSS）溶液を調製し、4％BSA、pH7.4中で、先に説明した3。 </li><li> 、150mMのLMEの最終濃度を得MEM培地5mlに純粋LMEを追加することによってLMEを調製する卓上pHメーターを用いてpH 7.4溶液を返し、フィルタ28ミリポリエーテルスルホン（PES）、0.2μmのシリンジを使用して滅菌フィルター。 </li></ol> 3. CGCSを培養<ol><li>以前に3を説明したように4-7日齢のラットの子から小脳を収集します。氷上で5ミリリットル溶液Aを含むペトリ皿に直ちに置きます。 </li><li>シャーレのふたで小脳と場所組織から余分な溶液を捨てます。 </li><li>少なくとも三つの異なる方向によく燃え上がっかみそりの刃で細かく組織をみじん切りに。 </li><li> 5分間37℃の水浴中で溶液（失われた制限された組織を確保するために溶液でペトリ皿をすすぐ）B及び場所にみじん切り組織を加えます。静か分おきに振ります。 </li><li>トリプシン、手ぶれやセントを中和するために、チューブに溶液D（20ミリリットル）を追加5分間、65×gでrifuge。 </li><li>上清を捨てます。 </li><li>直径を小さくする3燃え上がっガラスピペットで十分に溶液C.トリチュレート4mlの（約10回ずつ）で再懸濁し、懸濁液をできるだけ均一になるまで。 </li><li>ゆっくり、ゆっくりとBSA / EBSSの上にこのホモジネートを数滴を追加します。それはBSA / EBSSを通じてシンクを開始した場合は、より多くを粉砕するおよび/またはもう少し溶液Cを追加ホモジネートは、BSA / EBSSの上の層に座ってする必要があります。振らないでください。 5分間100×gでスピン。 </li><li>上清およびMEM培地1mlでペレットを再懸濁し（ソフト）を取り外します。 </li><li> MEM培地500μlの80万細胞/カバーガラスで血球計数器およびプレートを用いて細胞数を計測します。 6％CO 2で37℃のインキュベーターに入れ。 </li><li>培地を翌日（分取した後24〜36時間）を変更します。細胞周期阻害剤のアラビノフラノシルシチジン（のAraC）の10μMを含むMEM培地の溶液を作ります。 <ol><li>それぞれについてカバーガラスにして、再び細胞とトップまでの古い培地を250μlを追加、通常のMEM培地250μlを添加し、細胞を洗浄するために、これを吸引し、残りの部分を吸引、新鮮な24ウェルプレートに古い培地250μlのを保持のAraC含有培地250μlのと。 注意：細胞は、 インビトロ （DIV） で 6日間使用することができます。 </li></ol></li></ol> 4.を選択ミクログリアを枯渇（6 DIVに出演） <ol><li> 150mMのLMEの最終濃度が得られるようにMEM培地の別々のアリコートを5 mlに純粋なLMEを追加します。 </li><li> pHを7.4に解決策を戻し、フィルターは28ミリメートルのポリエーテルスルホン（PES）、0.2μmのシリンジフィルターを用いて滅菌します。 </li><li>コントロール培養のための通常のMEM培地と共に10分間37°Cの水浴中でMEM培地と場所での50mM（2×LME）の濃度に溶液を希釈。 </li><li> CGC培養から半分（250μl）をMEM培地を除去し、37℃で保持します。 </li><li>細胞を処理します、または対照培養のためのLMEのない予め温めたMEM培地（250μl）を、25 mMとウェル濃度を与える1： すなわち 2×LME（250μl）を含むMEM培地で1希釈しました。 </li><li> 1時間、6％CO 2の加湿雰囲気中、37℃で細胞をインキュベートします。 </li><li>洗浄細胞を2回新鮮予め温めたMEM培地中のLME含有培地を除去し、その後、対応するウェルに保持された培地と新鮮予め温めたMEM培地を等量置き換えます。 </li><li> （37℃で6％CO 2の加湿）インキュベーターに文化を戻し、任意のさらなる処理の前に24時間休ませるままにします。 </li></ol>

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The authors declare that they have no competing financial interests.

CGCからミクログリアの成功選択的除去を確実にするために最も重要なステップおよび/または混合培養物は、1）滅菌し、健康CGC文化を維持します。 2）フィルタは、LME含有培地を滅菌し、pHを7.4に溶液を戻します。 3）37に保持CGC媒体とLME含有培地を維持 ヒートショックを回避するため°C; 4）細胞をインキュベーターの外に保持される時間を短縮するために迅速に取り組ん。 <p class=...

人間の脳は、推定850億のニューロンおよびグリア1を含むさらに850億非神経細胞を含みます。過去100年間の大部分のために神経科学者は、ニューロンのための構造的支持を提供した受動支持細胞より少しであることがグリア細胞を信じて、神経細胞集団に主に焦点を当てている - それ故に「グリア」のギリシャ語の語源は、英語に翻訳します「接着剤」。しかし最近では、それは、神経膠細胞の相互作用がはるかに根本的な神経生物学、神経生理学、および多くの神経変性疾患の発生および進行の基本的な側面になる可能性があることがますます明らかになりました。小脳顆粒細胞（CGCS）、ヒトの脳で最も豊富な均質なニューロン集団は、小脳を支配し、その細胞成分の90％以上を占めています。したがって、これらの細胞は、Tのためのモデル系として、in vitroで広範に使用されています彼は、神経発達、機能、および病理2-6の検討します。 しかし、CGC培養はまだ間違いなくかなりの割合でミクログリア及びその他のグリアが含まれています。その結果、推定される種々の細胞治療への直接神経細胞応答を表示するCGCデータは、実際に発生する可能性がある - 一部または全体で - 文化の中で、隣接するグリアの間接的な二次応答から。これを評価するために、我々は、選択的に、L-ロイシンメチルエステル（LME）の助けを借りてCGCニューロン培養物からミクログリアを除去しました。 LMEは、もともと選択マクロファージ7を破壊するために使用lysomotropic剤であり、以降も選択的神経、アストロサイト、および混合膠細胞培養8,9,10からミクログリアを枯渇させるために使用されてきました。 LMEは、リソソームを破砕した後、アポトーシス13,14を引き起こしており、マクロファージおよびミクログリアによって内在化されます。マクロファージおよびミクログリアは、彼らがparticulaであることを引き起こして、リソソームにおける特徴豊富ですLMEの治療に暴露されるとRLY脆弱。このプロトコルは、CGC、その他のグリア/ニューロンの培養系を利用した実験でミクログリアの貢献を把握するための強力な、まだシンプルで簡単な方法を提供します。

<table><tbody><tr><td>Forceps&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F4142</td><td>The curved end facilitates removal of the cerebellum&amp;nbsp;</td></tr><tr><td>Micro-dissecting scissors</td><td>Sigma-Aldrich</td><td>S3146</td><td>Straight, sharp point facilitates rodent P4-7 dissection&amp;nbsp;</td></tr><tr><td>L-leucine methyl ester hydrochloride</td><td>Sigma-Aldrich</td><td>7517-19-3</td><td></td></tr><tr><td>EBSS solution&amp;nbsp;</td><td>Sigma-Aldrich</td><td>E7510-500 ml</td><td></td></tr><tr><td>Poly-D-lysine</td><td>Sigma-Aldrich</td><td>27964-99-4</td><td>Coat coverslips 1 day before use&amp;nbsp;</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich</td><td>A9418</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Sigma-Aldrich</td><td>P4417</td><td></td></tr><tr><td>DNase</td><td>Sigma-Aldrich</td><td>D5025</td><td></td></tr><tr><td>Soybean trypsin inhibitor&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T6414</td><td></td></tr><tr><td>Mouse anti-ED1 antibody&amp;nbsp;</td><td>Abcam</td><td>ab31630</td><td></td></tr></tbody></table>

selective depletion of microglia from cerebellar granule cell cultures using l-leucine methyl ester

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating innate immunity. Primary rodent cell culture models have greatly advanced our understanding of neuronal-glial interactions, but only recently have methods to specifically eliminate microglia from mixed cultures been utilized. One such technique &#8211; described here &#8211; is the use of L-leucine methyl ester, a lysomotropic agent that is internalized by macrophages and microglia, wherein it causes lysosomal disruption and subsequent apoptosis13,14. Experiments using L-leucine methyl ester have the power to identify the contribution of microglia to the surrounding cellular environment under diverse culture conditions. Using a protocol optimized in our laboratory, we describe how to eliminate microglia from P5 rodent cerebellar granule cell culture. This approach allows one to assess the relative impact of microglia on experimental data, as well as determine whether microglia are playing a neuroprotective or neurotoxic role in culture models of neurological conditions, such as stroke, Alzheimer&#8217;s or Parkinson&#8217;s disease.

選択的CGCおよび/または混合培養物からミクログリアを排除するために、この技術の能力を正確に識別し、その周囲の細胞からミクログリアを区別するために、研究者のその後の能力に依存しています。 図1に示されるように、これは、例えば、イソレクチンB4として、マイクログリア特異的細胞マーカーを使用して達成することができる。 図2に示すように、星状細?...

Watch this Scientific Journal Video about Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester at JoVE.com

Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester

Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature.

L-ロイシンメチルエステルを使用した小脳顆粒細胞培養物からのミクログリアの選択的枯渇

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating ...

selective-depletion-microglia-from-cerebellar-granule-cell-cultures

Research

JoVE Journal

Neuroscience

8.6K ビュー.  University of Washington. Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature. 

ビデオ: Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

新生仔ラットの脳組織の混合グリア細胞培養物から主要なミクログリアの分離

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

免疫・感染学

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-&#954;B (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-&#945; (TNF-&#945;), upon lipopolysaccharide (LPS) and Pam3CSK4 (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions.

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam3CSK4 (Pam).

マウス新生児から保護免疫表現と機能皮質ミクログリアの分離

Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure ...

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the co-existence of glial cells and neurons in CGN culture might lead to biases in the accurate assessment of neuronal viability. Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining has been used to measure cell viability by simultaneously evaluating the viable and dead cells. We used FDA-PI double staining to improve the sensitivities of the colorimetric assays and to evaluate neuronal viability in CGNs. Furthermore, we added blue fluorescent DNA stains (e.g., Hoechst) to improve the accuracy. This protocol describes how to improve the accuracy of assessment of neuronal viability by using these methods in CGN culture. Using this protocol, the number of glial cells can be excluded by using fluorescence microscopy. A similar strategy can be applied to distinguish the unwanted glial cells from neurons in various mixed cell cultures, such as primary cortical culture and hippocampal culture.

This protocol describes how to accurately measure neuronal viability using Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining in cultured cerebellar granule neurons, a primary neuronal culture used as an in vitro model in neuroscience and neuropharmacology research.

小脳顆粒ニューロン培養におけるFluorescein Diacetate-Propidium Iodide二重染色を用いたニューロン生存率の評価

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the ...

神経科学

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has revealed microglia to be dynamic, capable of assuming both pro-inflammatory and anti-inflammatory phenotypes. Both M1 (pro-inflammatory) and M2 (pro-reparative) phenotypes play an important role in neuroinflammatory conditions such as perinatal brain injury, and exhibit differing functions in response to certain environmental stimuli. The modulation of microglial activation has been noted to confer neuroprotection thus suggesting microglia may have therapeutic potential in brain injury. However, more research is required to better understand the role of microglia in disease, and this protocol facilitates that. The protocol described below combines a density gradient centrifugation process to reduce cellular debris, with magnetic separation, producing a highly pure sample of primary microglial cells that can be used for in vitro experimentation, without the need for 2-3 weeks culturing. Additionally, the characterization steps yield robust functional data about microglia, aiding studies to better our understanding of the polarization and priming of these cells, which has strong implications in the field of regenerative medicine.

A protocol for the isolation of primary microglia from murine brains is presented. This technique aids in furthering the current understanding of neurological conditions. Density gradient centrifugation and magnetic separation are combined to produce sufficient yield of a highly pure sample. Furthermore, we outline the steps for characterization of microglia.

特性と密度勾配遠心によるマウスのプライマリ ミクログリアの分離

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has ...

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive human diseases. Considerable advances have been made to uncover the molecular signatures of homeostatic microglia as well as alterations of their gene expression in response to environmental stimuli. With the advent and maturation of single-cell genomic methodologies, it is increasingly recognized that heterogenous microglia may underlie the diverse roles they play in different developmental and pathological conditions. Further dissection of such heterogeneity can be achieved through efficient isolation of microglia from a given region of interest, followed by sensitive profiling of individual cells. Here, we provide a detailed protocol for the rapid isolation of microglia from different brain regions in a single adult mouse brain hemisphere. We also demonstrate how to use these sorted microglia for plate-based deep single-cell RNA sequencing. We discuss the adaptability of this method to other scenarios and provide guidelines for improving the system to accommodate large-scale studies.

We provide a protocol for isolation of microglia from different dissected regions of an adult mouse brain hemisphere, followed by semi-automated library preparation for deep single-cell RNA sequencing of full-length transcriptomes. This method will help to elucidate functional heterogeneity of microglia in health and disease.

ディープシングルセルRNAシーケンシングのための1つの成人マウス脳半球からの領域特異的ミクログリアの分離

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive ...

Isolation of Mouse Primary Microglia by Magnetic-Activated Cell Sorting in Animal Models of Demyelination

Microglia, the resident innate immune cells in the brain, are the primary responders to inflammation or injury in the central nervous system (CNS). Microglia can be divided into resting state and activated state and can rapidly change state in response to the microenvironment of the brain. Microglia will be activated under different pathological conditions and exhibit different phenotypes. In addition, there are many different subgroups of activated microglia and great heterogeneity between different subgroups. The heterogeneity mainly depends on the molecular specificity of microglia. Studies have revealed that microglia will be activated and play an important role in the pathological process of inflammatory demyelination. To better understand the characteristics of microglia in inflammatory demyelinating diseases such as multiple sclerosis and neuromyelitis optica spectrum disorder, we propose a perilesional primary microglial sorting protocol. This protocol utilizes columnar magnetic-activated cell sorting (MACS) to obtain highly purified primary microglia and preserve the molecular characteristics of microglia to investigate the potential effects of microglia in inflammatory demyelinating diseases.

Here, we present a protocol to isolate and purify primary microglia in animal models of demyelinating diseases, utilizing columnar magnetic-activated cell sorting.

脱髄の動物モデルにおける磁気活性化細胞選別によるマウス原発性ミクログリアの単離

Microglia, the resident innate immune cells in the brain, are the primary responders to inflammation or injury in the central nervous system (CNS). ...

Modeling Reactive Oxygen Species-Induced Neuronal Death in Mouse Cerebellar Granule Neurons

Source: Laaper, M. et al., <a href="https://app.jove.com/v/55871">Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons.</a>&#160;J. Vis. Exp. (2017).
This video outlines the generation of a neuronal injury model using reactive oxygen species (ROS) in mouse primary cerebellar granule neurons. It details the process of inducing oxidative stress by adding high concentrations of hydrogen peroxide. ROS exposure causes DNA strand breaks and genomic instability. Additionally, the oxidation of cellular proteins impairs their functions, while lipid peroxidation compromises membrane integrity. This cascade leads to cytochrome c release and caspase activation, resulting in neuronal apoptosis.

マウス小脳顆粒ニューロンにおける活性酸素種誘発性ニューロン死のモデル化

1. Experimental Preparation
NOTE: The following stock solutions can be prepared and maintained until use.

	Dissection Solution
	
		Dissolve 3.62 g of ...

JoVE Encyclopedia of Experiments

Neuropathology

Neurodegenerative Diseases

Isolation of Microglia from the Cortex of an Adult Mouse Brain

Source: Zhou, L., et al., <a href="https://app.jove.com/v/60347">Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing.</a> J. Vis. Exp. (2019)
The video demonstrates a technique for isolating microglia from the cortex of an adult mouse brain. First, the cortex undergoes segmentation and homogenization to remove neurons and other glial cells, preserving microglia. Following that, the cell suspension is exposed to anti-myelin magnetic microbeads and a magnetic field, effectively separating microglia and myeloid cells from myelin debris and cells containing myelin.

成体マウス脳の皮質からのミクログリアの分離

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Mechanical Tissue Dissociation
...

神経細胞培養技術

一次神経細胞培養

Isolation and Culture of Mouse Primary Cerebellar Granule Neurons

Source: Laaper, M., et al. <a href="https://app.jove.com/v/55871">Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons.</a> J. Vis. Exp. (2017)
This video demonstrates the technique for isolating and culturing primary mouse cerebellar granule neurons. Cells are treated with an antimetabolite to eliminate proliferating glial cells and produce a pure culture of cerebellar granule neurons.

マウス初代小脳顆粒ニューロンの単離と培養

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Experimental Preparation
NOTE: ...

Selectively Removing Microglia from a Rat Cerebellar Granule Neuron Culture

Source: Jebelli, J., et al. <a href="https://app.jove.com/v/52983">Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester.</a> J. Vis. Exp. (2015)
This video demonstrates a technique for the selective removal of microglia from a primary rat neuronal culture. Cerebellar granule neurons are treated with an antimetabolite to eliminate proliferating glial cells. Additionally, they are treated with lysosome-targeting agents to deplete microglia from the culture.

ラット小脳顆粒ニューロン培養物からのミクログリアの選択的除去

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of Instruments, ...

L-ロイシンメチルエステルを使用した小脳顆粒細胞培養物からのミクログリアの選択的枯渇

要約

さらに動画を探す

この動画の章

新生仔ラットの脳組織の混合グリア細胞培養物から主要なミクログリアの分離

マウス新生児から保護免疫表現と機能皮質ミクログリアの分離

小脳顆粒ニューロン培養におけるFluorescein Diacetate-Propidium Iodide二重染色を用いたニューロン生存率の評価

特性と密度勾配遠心によるマウスのプライマリミクログリアの分離

ディープシングルセルRNAシーケンシングのための1つの成人マウス脳半球からの領域特異的ミクログリアの分離

脱髄の動物モデルにおける磁気活性化細胞選別によるマウス原発性ミクログリアの単離

マウス小脳顆粒ニューロンにおける活性酸素種誘発性ニューロン死のモデル化

成体マウス脳の皮質からのミクログリアの分離

マウス初代小脳顆粒ニューロンの単離と培養

ラット小脳顆粒ニューロン培養物からのミクログリアの選択的除去

L-ロイシンメチルエステルを使用した小脳顆粒細胞培養物からのミクログリアの選択的枯渇

要約

さらに動画を探す

この動画の章

新生仔ラットの脳組織の混合グリア細胞培養物から主要なミクログリアの分離

マウス新生児から保護免疫表現と機能皮質ミクログリアの分離

小脳顆粒ニューロン培養におけるFluorescein Diacetate-Propidium Iodide二重染色を用いたニューロン生存率の評価

特性と密度勾配遠心によるマウスのプライマリ ミクログリアの分離

ディープシングルセルRNAシーケンシングのための1つの成人マウス脳半球からの領域特異的ミクログリアの分離

脱髄の動物モデルにおける磁気活性化細胞選別によるマウス原発性ミクログリアの単離

マウス小脳顆粒ニューロンにおける活性酸素種誘発性ニューロン死のモデル化

成体マウス脳の皮質からのミクログリアの分離

マウス初代小脳顆粒ニューロンの単離と培養

ラット小脳顆粒ニューロン培養物からのミクログリアの選択的除去

特性と密度勾配遠心によるマウスのプライマリミクログリアの分離