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著者が明らかに何もありません。

ミクログリアは、環境刺激によって変更両方 pro - と抗炎症、能力を持っています。前の研究は、ミクログリアの活性化の変調は神経保護を与えることができる示されています。ニューロンに保護を提供して損傷を修復する能力は、これらの複雑な細胞の現在の理解を促進するためのより多くの研究を必要とします。したがって、高純度プライマリ ミクログリアの分離は重要かつ有用な手法?...

炎症、低酸素虚血および出血から周産期の期間中に取得した損傷は長期的後遺症の配列を持つことができます。周産期の脳損傷の複雑な病態は炎症や虚血後の神経細胞と軸索の死1理論します。生得の免疫反応は、傷害2につながるイベントの連鎖で重要な役割を果たしています。
ミクログリア、中枢神経系 (CNS)、内居住者の免疫細胞は、傷害3の最初の反応です。ミクログリアは、4の環境保護や毒性、依存する容量を持つプラスチック電池種類です。走化性、食作用、抗原提示、サイトカインや活性酸素種4、5の生産に関与しています。老齢ミクログリアは常に環境を調査し、外国または有害物質4の存在によってアクティブ化されます。活性化は、中枢神経系の保護4で重要な炎症応答に します。これらの M1「炎症」表現型ミクログリア主に抗原提示と病原体4の死に関与しています。にもかかわらず、神経保護の炎症反応の重要な役割は、自由なまたは長期の炎症が神経損傷4につながる有害なにできます。ただし、特定の環境の刺激にさらされると、ミクログリアは抗炎症の表現型を表わすことができます。これら pro 修復 M2 ミクログリア創傷治癒と修復6各種のサイトカインを放出する重要な役割があるし、他の溶性メディエーター レセプター炎症、貪食能を増加し、促進する修復4, 7. ミクログリアの役割は多様であり、再髄鞘化8、脳卒中モデル9で酸素とグルコース枯渇中に神経細胞を保護して神経突起を推進運転オリゴデンドロ サイト分化脊髄損傷は、10をモデル化します。
これらのグリア細胞の研究を理解および neuroinflammation への応答の処理で重要な側面を表します。記述のプロトコル アミロイド疾患におけるミクログリアの変調方式の治療の可能性をさらに調査できます。
ミクログリア活性化神経の役割への変調は、条件11,12,13の範囲で観察されています。したがって、現在の理解と活性化ミクログリアの変調をさらに勉強して改善は重要なin vitroとin vivoの両方を含むさまざまなモデルの使用を必要とします。In vitroの研究より効率、低いコストと能力孤立セル人口を調査するための重要なツールを表します。
ミクログリア マウス脳から効率的に良い生存率および高純度と高収率のサンプルを生成する課題の分離のための文献に記載されているプロトコルの範囲があります。プライマリ ミクログリアの分離の一般的に使用される方法は、磁気分離と混合培養の揺れです。個人的な経験を通して、磁気の列を妨げ細胞の残骸の高度があったことが分られました。したがって、次のプロトコルは、CD11b 磁気分離に続いて初期密度勾配遠心法手順を組み込んだに利用されました。下記プロトコルは、十分な量の非常に純粋なサンプルを生成する最適化されています。その高純度と短い期間のために有利である-1 つは、2-3 週間培養することがなく 2 日以内の試金を実行できます。このプロトコルは、プライマリ マウスアストロの隔離のため潜在的適応ことができます。

<table><tbody><tr><td>DMEM, low glucose, pyruvate</td><td>Gibco</td><td>11885084</td><td></td></tr><tr><td>Antibiotic-Antimycotic (100X)</td><td>Gibco</td><td>15240062</td><td></td></tr><tr><td>DNaseI grade II from bovine pancreas</td><td>Sigma-Aldrich</td><td>10104159001</td><td></td></tr><tr><td>Papain from papaya latex, buffered aqeuous solution</td><td>Sigma-Aldrich</td><td>P3125-100mg</td><td></td></tr><tr><td>Fetal Bovine Serum, qualified, heat inactivated</td><td>Gibco</td><td>16140071</td><td></td></tr><tr><td>Percoll</td><td>GE Healthcare</td><td>17-0891-01</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution (1X)</td><td>Gibco</td><td>14175-103</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution (10X)</td><td>Gibco</td><td>14185052</td><td></td></tr><tr><td>EasySep Mouse CD11b Positive Selection Kit</td><td>StemCell Technologies</td><td>18770</td><td>EasySep magnet variant</td></tr><tr><td>EasySep magnet</td><td>StemCell Technologies</td><td>18000</td><td></td></tr><tr><td>EasySep Buffer</td><td>StemCell Technologies</td><td>20144</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate buffered saline</td><td>Gibco</td><td>14040182</td><td></td></tr><tr><td>Trypsin (2.5%) (10X)</td><td>Gibco</td><td>15090-046</td><td></td></tr><tr><td>Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block&amp;trade;)</td><td>BD Biosciences</td><td>553141</td><td></td></tr><tr><td>Falcon 5mL Round Bottom High Clarity PP Test Tube, with Snap Cap, Sterile</td><td>Corning</td><td>352063</td><td></td></tr><tr><td>175cm&amp;sup2; Angled Neck Cell Culture Flask with Vent Cap</td><td>Corning</td><td>431080</td><td></td></tr><tr><td>Lipopolysaccharides from Escherichia coli O127:B8</td><td>Sigma-Aldrich</td><td>L5024</td><td></td></tr><tr><td>96 Well TC-Treated Microplates size 96 wells, clear, polystyrene, round bottom</td><td>Corning</td><td>CLS3799</td><td></td></tr><tr><td>Paraformaldehyde (powder, 95%)</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>Triton-X</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>Rabbit Anti-Iba1</td><td>Wako</td><td>01919741&amp;nbsp;</td><td></td></tr><tr><td>Goat Anti-Rabbit IgG H&amp;amp;L (Alexa Fluor 488)</td><td>Abcam</td><td>ab150077</td><td></td></tr><tr><td>&lt;strong&gt;FACS Antibodies&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td></td></tr><tr><td>V450,Rat,Anti-Mouse,CD45,30-F11,RUO</td><td>BD Biosciences</td><td>560501</td><td></td></tr><tr><td>PerCP-Cy5.5 CD11b&amp;nbsp;</td><td>eBiosciences</td><td>45-0112-82</td><td></td></tr><tr><td>ZombieNIR</td><td>Biolegend</td><td>423105</td><td></td></tr><tr><td>pHrodo Red E. coli BioParticles Conjugate</td><td>Thermo Fisher Scientific</td><td>P35361</td><td></td></tr><tr><td>Annexin.V_FITC</td><td>Miltenyi Biotech</td><td>130-093-060</td><td></td></tr><tr><td>Propodium Iodide solution</td><td>Miltenyi Biotech</td><td>130-093-233</td><td></td></tr></tbody></table>

characterization and isolation of mouse primary microglia by density gradient centrifugation

ミクログリア、脳内の居住者の免疫細胞は、炎症や中枢神経系に傷害の最初の反応です。最近の研究は、動的、プロ炎症性と抗炎症の表現型を想定しての対応するミクログリアを明らかにしました。M1 (炎症) と M2 (pro 修復) 表現型プレイ アミロイド条件周産期の脳損傷など展示異なる重要な役割機能ある特定の環境刺激に応答します。ミクログリア活性化の変調は神経保護ミクログリアが脳損傷の治療の可能性あるかもしれませんが示唆を与えるに指摘されています。しかしより多くの研究は病気におけるミクログリアの役割を理解するために必要なこのプロトコルを容易にします。プロトコルは下記を組み合わせた磁気分離、体外実験のために使用できる主要な小グリア細胞の純度の高いサンプルの生産と細胞の残骸を減らすために密度勾配の遠心分離プロセス、2-3 週間培養の必要があります。さらに、評価手順は、ミクログリア、偏光の私達の理解を改善するための研究を支援し、これらの細胞のプライミング再生医療の分野で強力な影響を持っている情報の堅牢な機能を得られます。

ここで説明した方法を使用して、ミクログリアの純粋な人口は分離できるので、体外と FACS 解析を用いた特性評価のために準備することができます。そもそも、18 動物まで使用できます選別、約 450,000 600,000 ミクログリア細胞の期待利回りあたり。最初に単離細胞の純度を確認することが重要だし、CD45 と CD11b ミクログリアの同定、2 つのマーカーの染色で FACS ...

Watch this Scientific Journal Video about Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation at JoVE.com

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

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

マウスの脳からプライマリ ミクログリアの隔離のためのプロトコルが表示されます。この手法は、神経学的な条件の現在の理解の促進に役立ちます。密度勾配遠心法と磁気分離は、純度の高いサンプルの十分な収量を生成する結合されます。さらに、ミクログリアの特性評価のための手順をまとめました。

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

characterization-isolation-mouse-primary-microglia-density-gradient

Research

JoVE Journal

Neuroscience

19.1K ビュー.  Hudson Institute of Medical Research. マウスの脳からプライマリ ミクログリアの隔離のためのプロトコルが表示されます。この手法は、神経学的な条件の現在の理解の促進に役立ちます。密度勾配遠心法と磁気分離は、純度の高いサンプルの十分な収量を生成する結合されます。さらに、ミクログリアの特性評価のための手順をまとめました。

ビデオ: Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

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 ...

免疫・感染学

Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. Microglia are mesodermal cells that function similarly to macrophages in surveying inflammatory stress. The classical (M1-type) and alternative (M2-type) activations of macrophages have also been extended to microglia in an effort to better understand the underlying interplay these phenotypes have in neuroinflammatory conditions such as Parkinson&#39;s, Alzheimer&#39;s, and Huntington&#39;s Diseases. In vitro experimentation utilizing primary microglia offers rapid and reliable results that may be extended to the in vivo environment. Although this is a clear advantage over in vivo experimentation, isolating microglia while achieving adequate yields of optimal purity has been a challenge. Common methods currently in use either suffer from low recovery, low purity, or both. Herein, we demonstrate a refinement of the column-free CD11b magnetic separation method that achieves a high cell recovery and enhanced purity in half the amount of time. We propose this optimized method as a highly useful model of primary microglial isolation for the purposes of studying neuroinflammation and neurodegeneration.

Here, we present a protocol to isolate microglia from postnatal mouse pups (day 1) for in vitro experimentation. This improvised method of isolation generates both high yield and purity, a significant advantage over alternate methods that allows broad range experimentation for the purposes of elucidating microglial biology.

強化された純度と汎用性を持つ初代ミクログリアの迅速かつ洗練されたCD11b磁気分離

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. ...

神経科学

Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium

Microglia represent 5 - 10% of all central nervous system (CNS) cells and are increasingly drawing attention due to their contributions during development, homeostasis, and disease. Although macrophages have been studied in detail for decades, specialized features of microglia, the tissue-resident macrophages of the CNS, have remained largely mysterious, in part due to limitations in the ability to recapitulate mature microglial properties in culture. Here, we illustrate a straightforward procedure for the rapid isolation of pure microglia from the mature rodent brain. We also describe serum-free culture conditions that support high levels of microglial viability over time. Microglia cultured under these defined-medium conditions exhibit elaborate ramified processes and dynamic surveillance behavior. We illustrate some effects of serum exposure on cultured microglia and discuss how these serum-free cultures compare to both serum-exposed cultures as well as microglia in vivo.

Efforts to understand microglial function in detail have been hindered by the lack of microglial culture models that recapitulate the properties of mature in vivo microglia. This protocol describes an isolation and culture approach designed to maintain robust survival of highly ramified mature rat microglia under defined-medium conditions.

動的なを促進するために齧歯動物のミクログリアの分離と培養分岐無血清培地の形態

Microglia represent 5 - 10% of all central nervous system (CNS) cells and are increasingly drawing attention due to their contributions during ...

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 ...

Primary Microglia Isolation from Postnatal Mouse Brains

Microglia are the mononuclear phagocytes in the central nervous system (CNS), which play key roles in maintaining homeostasis and regulating the inflammatory process in the CNS. To study the microglial biology in vitro, primary microglia show great advantages compared to immortalized microglial cell lines. However, microglia isolation from the postnatal mouse brain is relatively less efficient and time-consuming. In this protocol, we provide a quick and easy-to-follow method to isolate primary microglia from the neonatal mouse brain. The overall steps of this protocol include brain dissection, primary brain cell culture, and microglia isolation. Using this approach, researchers can obtain primary microglia with high purity. In addition, the harvested primary microglia were able to respond to the lipopolysaccharides challenge, indicating they retained their immune function. Collectively, we developed a simplified approach to efficiently isolate primary microglia with high purity, which facilitates a wide range of microglial biology investigations&#160;in vitro.

Primary cell culture is one of the primarily used approaches for studying microglial biology in vitro. Here, we developed a method for simple and rapid microglia isolation from the mouse postnatal day 1 (P1) to P4.

Microglia are the mononuclear phagocytes in the central nervous system (CNS), which play key roles in maintaining homeostasis and regulating the ...

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). ...

In vitroアッセイのためのMACSを用いたマウスからの視床下部ミクログリアの単離

Isolation of Hypothalamic Microglia from Freshly Perfused Adult Mouse Brain by Magnetic-Activated Cell Sorting

Microglia, as the resident macrophages of the brain, are essential for maintaining brain homeostasis. They shape neuronal circuits during development, survey their environment for debris or dead cells, as well as respond to infection and injury in the brain, among many other functions. However, their important role in neurodevelopment and synaptic plasticity and pathophysiology has not been fully defined, highlighting the need for further investigation. To gain a more comprehensive understanding of the role of microglia in these processes, we need to isolate microglia and characterize them genetically, metabolically, and functionally. However, the isolation of microglia from adult mice, especially from small brain structures, is challenging as they represent a small percentage of the total brain cells, and the yield of isolated microglia is often too low. Here, the magnetic isolation of microglia using CD11b+ microbeads allows us to sort microglial cells from the hypothalamus of a freshly perfused adult mouse brain. The current method allows us to achieve relatively high purity and yield in a short period while maintaining cell viability.

著者スポットライト:マウスの脳機能および代謝障害に対する栄養の影響の調査 

We describe a protocol for isolating microglial cells from the mouse hypothalamus (or equivalent small brain structures) using magnetically activated cell sorting (MACS), in a relatively short time. The MACS-sorted hypothalamic microglia can be used for ex vivo analysis and can be plated to perform in vitro assays.

磁気活性化細胞選別による灌流したばかりの成体マウス脳からの視床下部ミクログリアの単離  

Microglia, as the resident macrophages of the brain, are essential for maintaining brain homeostasis. They shape neuronal circuits during development, ...

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
...

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

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この動画の章

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

強化された純度と汎用性を持つ初代ミクログリアの迅速かつ洗練されたCD11b磁気分離

動的なを促進するために齧歯動物のミクログリアの分離と培養分岐無血清培地の形態

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

Primary Microglia Isolation from Postnatal Mouse Brains

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

磁気活性化細胞選別による灌流したばかりの成体マウス脳からの視床下部ミクログリアの単離

磁気活性化細胞選別による灌流したばかりの成体マウス脳からの視床下部ミクログリアの単離

磁気活性化細胞選別による灌流したばかりの成体マウス脳からの視床下部ミクログリアの単離

成体マウス脳の皮質からのミクログリアの分離

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

要約

さらに動画を探す

この動画の章

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

強化された純度と汎用性を持つ初代ミクログリアの迅速かつ洗練されたCD11b磁気分離

動的なを促進するために齧歯動物のミクログリアの分離と培養分岐無血清培地の形態

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

Primary Microglia Isolation from Postnatal Mouse Brains

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

磁気活性化細胞選別による灌流したばかりの成体マウス脳からの視床下部ミクログリアの単離

磁気活性化細胞選別による灌流したばかりの成体マウス脳からの視床下部ミクログリアの単離

磁気活性化細胞選別による灌流したばかりの成体マウス脳からの視床下部ミクログリアの単離

成体マウス脳の皮質からのミクログリアの分離

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