この研究は、スウェーデンの研究評議会（K2008 - 57X - 20 676-01-3）によって賄われた。

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+exosomes+expressing+Fas+ligand+mediate+CD8++T-cell+apoptosis.">Tumor exosomes expressing Fas ligand mediate CD8+ T-cell apoptosis.</a> Blood Cells, Molecules, and Diseases. 35, 169-173 (2005).</li><li>Valadi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosome-mediated+transfer+of+mRNAs+and+microRNAs+is+a+novel+mechanism+of+genetic+exchange+between+cells.">Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.</a> Nat. Cell Biol. 9, 654-659 (2007).</li><li>Eldh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+communicate+protective+messages+during+oxidative+stress;+possible+role+of+exosomal+shuttle+RNA.">Exosomes communicate protective messages during oxidative stress; possible role of exosomal shuttle RNA.</a> PLoS One. 5, e15353-e15353 (2010).</li><li>L&#228;sser, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+saliva,+plasma+and+breast+milk+exosomes+contain+RNA:+uptake+by+macrophages.">Human saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages.</a> J. Transl. Med. 9, 9-9 (2011).</li></ol>

JLは、細胞へのRNAの転送のためのベクターとしてエクソソームの利用に関連する特許の共同所有者です。

分子コンテンツおよび/またはエクソソームの機能を研究するとき、それは適切な収率と汚染の可能な限り低い学位を提供する高品質の単離法を使用することが重要です。このホワイトペーパーで説明されている方法論は、エクソソームを分離し、それらのRNA含有量と生物学的機能を研究するための十分に確立されたアプローチです。さらに、孤立エクソソームの特性評価の重要性は、電子顕微鏡、フローサイトメトリー、ウェスタンブロットを含むさまざまな方法の組み合わせによって、強調表示されます。 エクソソームを分離する場合、最初の遠心分離工程（300 × gでは）細胞懸濁液または調査生物学的流体中に存在する可能性のある細胞をペレット化するために使用されます。ペレット死細胞、大きい破片、アポトーシス小体や他の細胞小器官に十分に強いものに16 500 × gでのニーズの第二遠心分離。この遠心分離の後、一部のプロトコルは、ナノろ過工程が、いくつかINVが含まれていますestigatorsは、このステップを除外している。しかし、我々は200nmよりも大きいの根絶の断片と小胞の重要性を強調するため、強く、濾過工程を含めることをお勧めします。より厳格な分離が必要な場合は、100nmのフィルターが使用されますが、高粘性流体が使用されている場合、これらのフィルタを簡単にブロックになることができるとexosomal材料が失われることに注意することができます。また、100nmのろ過は、エクソソームの形態に影響を与える可能性がありますし、さらにエクソソームが大規模にある場合、それらが失われる可能性があります。従って、200nmのフィルターは、エキソソーム分離に適しているようです。濾過後、120,000 xgで必須の超遠心分離は、ペレットエクソソームするために使用されます。エクソソームは、さらに抗体被覆ビーズに結合し、洗浄し、PBSで洗浄し、または汚染のタンパク質を除去するために、ショ糖勾配上に配置することができます。しかし、これはスタートサンプル量が小さいと/またはRNA含有量Oの場合は特に、我々は必要ではないと主張することは非常にリソースがかかる可能性がありますFエクソソームは、余分な手順は、実質的にさらに材料を減らすので、低いです。しかし、再び、隔離された小胞の性質は、特定のタンパク質の存在下および非存在によって証明されるべきである。 日付に、エクソソームには絶対的にユニークなタンパク質マーカーは、サンプルがエクソソーム、それ以外のものが含まれていることを確認するために同定されていない。その結果、方法の組み合わせは、電子顕微鏡によるエクソソームの大きさと形態の決定を含むエクソソームを、特徴づけるために必要とされています。このメソッドはそのような微粒子、アポトーシス小体や細胞の破片などの大規模な小胞、と例えば、サンプルの汚染のレベルの概要を提供することができるとしても、サンプルの純度は、電子顕微鏡によって測定することができる。また、エクソソームのタンパク質含有量（例えばCD63を結合した​​膜の両方の分析でフローサイトメトリーとウェスタンブロット法、及びこれら二つのメソッドの結果の組み合わせによって決定することができるとCD81）とエクソソームの内部化タンパク質（例えばTsg101とアリックス）。このようなCD63、Tsg101とアリックス、そしてそのような小胞体蛋白質カルネキシンのようなタンパク質の欠如など、エクソソーム、に富むタンパク質の検出は、エキソソーム濃縮ペレットが実際にエクソソームと細胞の他の区画からではない汚染する小胞であることを示しています。 エクソソームで検出されるRNAのプロファイルは、無傷の細胞で検出されたRNAのそれと根本的に異なっている。従って、バイオアナライザでまたはゲルベースの​​RNAの解析手法で分析したexosomal RNAは、細胞のRNAの解析において顕著な18Sと28S rRNAのサブユニット、の2つのピークを欠いている。したがって、サンプルのrRNAのいずれかのかなりの量の検出は、分離の方法が改善し、品質を保証する必要があることをこのように抽出したtotal RNAは単独でexosomal起源ではないことを示す、と主張する。

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（省略可能） </td></tr><tr><td> エキソソーム分離 </td></tr><tr><td>オプティマL - 90K超遠心機</td><td>ベックマンコールター</td><td> 65670 </td><td></td></tr><tr><td>クイックシールの超遠心管</td><td>ベックマンコールター</td><td></td><td>別のチューブを使用するローターに応じて必要とされる。 Ti70（342414）、Ti45（345776） </td></tr><tr><td>クイックシールコードレスチューブトッパーキット</td><td>ベックマンコールター</td><td> 358312 </td><td></td></tr><tr><td> Filtropurシリンジフィルター、0.20μm </td><td>ザルスタット</td><td> 83.1826.001 </td><td> VWRインターナショナルからビスコース液体および/または大容量、真空ろ過システム、0.2μmの、のために（514〜0600）の代わりに使用することができます。 </td></tr><tr><td> 電子顕微鏡法 </td></tr><tr><td> Formvar /カーボンコーティングされたニッケルグリッド</td><td>テッドペラ株式会社</td><td> 1GN200 </td><td></td></tr><tr><td>マウス抗ヒトCD63 </td><td>バイオサイエンスBD </td><td> 556019 </td><td>最終濃度：0.05μg/μl </td></tr><tr><td>アイソタイプコントロール、IgG1κ（MOPC 21） </td><td>シグマアルドリッチ</td><td> M9269 </td><td>最終濃度：0.05μg/μl </td></tr><tr><td>抗マウスIgG金共役、10nmの</td><td>シグマアルドリッチ</td><td> G7777 </td><td>最終濃度：1％ </td></tr><tr><td> LEO 912ABオメガ電子顕微鏡</td><td>カールツァイスNTS </td><td></td><td>または同等の装置。 </td></tr><tr><td> フローサイトメトリー </td></tr><tr><td> 4μmaldehyd /硫酸塩ラテックスビーズ</td><td>界面動力学</td><td> 12から4000 </td><td></td></tr><tr><td>ラテックスビーズのコーティング用抗体</td><td></td><td></td><td>バイオサイエンスBD（556019）からの例の抗CD63用</td></tr><tr><td>インテリミキサーRM - 2L </td><td> ELMI </td><td></td><td>または同等の装置。 </td></tr><tr><td>ヒト血清からのIgG </td><td>シグマアルドリッチ</td><td> I4506 </td><td></td></tr><tr><td>検出用抗体</td><td>バイオサイエンスBD </td><td></td><td>たとえば、次のようにPE抗CD63（556020）PE抗CD9（555372）PE、抗CD81（555676）アイソタイプコントロール（555749） </td></tr><tr><td> BD FACSAria </td><td>バイオサイエンスBD </td><td></td><td></td></tr><tr><td> ウエスタンブロット法 </td></tr><tr><td> Transsonic T 490 DH </td><td> ELMA </td><td></td><td>または同等の装置。 </td></tr><tr><td> exosomal RNAとRNA解析の分離 </td></tr><tr><td> miRCURY RNAアイソレーションキット</td><td> Exiqon A / S </td><td> 300110 </td><td></td></tr><tr><td> Agilentの2100バイオアナライザ</td><td>アジレントテクノロジー</td><td> G2939A </td><td></td></tr></tbody></table>

isolation and characterization of rna-containing exosomes

エキソソーム研究の分野は近年急速に出版物の劇的な増加と、拡大しています。エンドサイトーシスの起源のこれらの小胞は（30〜100 nmの）最初の赤血球1に成熟しながら、網状赤血球は、トランスフェリン受容体を根絶するための手段として機能するように提案し、後で名前エクソソームであった。エキソソームは、多胞体（MVBs）の製造、後期エンドソームの内向きの出芽によって形成され、原形質膜2 MVBsの融合により、環境中に放出されています。エクソソームの最初の発見以来、セルの広い範囲は、これらの小胞を放出することが示されている。エキソソームは、プラズマ、鼻洗浄液、唾液や母乳3月6日を含むいくつかの生物学的流体、検出されている。さらに、それはエクソソームの内容と機能は元の細胞とそれらが生成される条件に依存することが実証されている。様々な機能が悪魔となっているT細胞増殖14の刺激、制御性T細胞にそのようなアレルゲン7,8に対する寛容の誘導など、エクソソーム、、マウス9の確立された腫瘍の撲滅、ナチュラルキラー細胞10-12の抑制と活性化、分化の促進13のtratedとT細胞のアポトーシス15の誘導。 2007年に我々は、肥満細胞から放出さエクソソームは、メッセンジャーRNA（mRNA）とマイクロRNA（miRNAを）含んでいる、とRNAが1つのセルからエクソソームを経由して別のに往復することができるということを示した。レシピエントの細胞では、エクソソームで往復mRNAを転送RNA 16の調節機能を示唆し、タンパク質に翻訳されることが示された。さらに、我々はまた、酸化ストレス下で増殖した細胞から派生したエクソソームは、レシピエント細胞にさらなるストレスに対する耐性を誘導するため、exosomalシャトルRNA 17の生物学的機能を提案できることが示されている。細胞培養培地や生体液のCO小胞の混合物をntainとフラグメントを流した。エクソソームのための高品質の単離法は、特性評価およびエクソソームとそのコンテンツの識別に続いて、したがって、他の小胞と粒子からエクソソームを区別するために不可欠です。ここで、我々は、細胞培養の培地や体液の両方からエクソソームの単離のための方法を提示する。この分離法はエクソソームはペレット化されている最後の超遠心分離のステップに続いて、繰り返しの遠心分離やろ過のステップに基づいています。エクソソームを特定し、exosomal形態及び蛋白質含量を特徴づける重要なメソッドは、電子顕微鏡、フローサイトメトリーとウェスタンブロット法を含めて、強調表示されます。トータルexosomal RNAの精製はスピンカラムクロマトグラフィーに基づいており、exosomal RNAの収量とサイズ分布は、バイオアナライザを用いて分析される。

Watch this Scientific Journal Video about Isolation and Characterization of RNA-Containing Exosomes at JoVE.com

Isolation and Characterization of RNA-Containing Exosomes

本論文では、エクソソームの単離、精製および検出のための方法、ならびにそれらの分子量の分析のためのテクニックを示しています。これらのメソッドは、細胞培養液や体液の両方からエキソソーム分離のための適応であり、分子量の分析を越えても機能的研究に役立ちます。

RNA含有エキソソームの単離と解析

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

isolation-and-characterization-of-rna-containing-exosomes

Research

JoVE Journal

Biology

99.1K ビュー.  University of Gothenburg. 本論文では、エクソソームの単離、精製および検出のための方法、ならびにそれらの分子量の分析のためのテクニックを示しています。これらのメソッドは、細胞培養液や体液の両方からエキソソーム分離のための適応であり、分子量の分析を越えても機能的研究に役立ちます。

ビデオ: Isolation and Characterization of RNA-Containing Exosomes

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be produced to contain any desired DNA or RNA sequence and can be prepared to include a wide variety of base and sugar modifications. Moreover, oligos can be designed to mimic specific nucleic acid alterations and thus, can serve as important tools to investigate effects of DNA damage and mechanisms of repair. We found that Thermo Scientific Dharmacon RNA-containing oligos with a length between 50 and 80 nucleotides can be particularly suitable to study, in vivo, functions and consequences of chromosomal RNA/DNA hybrids and of ribonucleotides embedded into DNA. RNA/DNA hybrids can readily form during DNA replication, repair and transcription, however, very little is known about the stability of RNA/DNA hybrids in cells and to which extent these hybrids can affect the genetic integrity of cells. RNA-containing oligos, therefore, represent a perfect vector to introduce ribonucleotides into chromosomal DNA and generate RNA/DNA hybrids of chosen length and base composition. Here we present the protocol for the incorporation of ribonucleotides into the genome of the eukaryotic model system yeast /Saccharomyces cerevisiae/. Yet, our lab has utilized Thermo Scientific Dharmacon RNA-containing oligos to generate RNA/DNA hybrids at the chromosomal level in different cell systems, from bacteria to human cells.

This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.

RNAを含むオリゴヌクレオチドを​​使用して変換によるゲノムDNAのRNA / DNAハイブリッドの世代

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

生物学

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.

Isolation &amp; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

ヘキストの分離および特性評価低 CD45負マウス肺間葉系幹細胞

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

アイソレーション、文化、および成体マウスCardiomyoctyesの機能解析

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

Isolation and Characterization of Microvesicles from Peripheral Blood

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived microvesicles (MVs, diameter &#62; 100 nm) is a fundamental cellular process that occurs in all living cells. These vesicles transport proteins, lipids and nucleic acids specific for their cell of origin and in vitro studies have highlighted their importance as mediators of intercellular communication. EVs have been successfully isolated from various body fluids and especially EVs in blood have been identified as promising biomarkers for cancer or infectious diseases. In order to allow the study of MV subpopulations in blood, we present a protocol for the standardized isolation and characterization of MVs from peripheral blood samples. MVs are pelleted from EDTA-anticoagulated plasma samples by differential centrifugation and typically possess a diameter of 100 - 600 nm. Due to their larger size, they can easily be studied by flow cytometry, a technique that is routinely used in clinical diagnostics and available in most laboratories. Several examples for quality control assays of the isolated MVs will be given and markers that can be used for the discrimination of different MV subpopulations in blood will be presented.

Extracellular vesicles present in blood have been suggested as novel biomarkers for various diseases. Here, we present a protocol for the isolation of large plasma membrane-derived microvesicles from peripheral blood samples and their subsequent analysis by conventional flow cytometry and Western Blotting.

末梢血からの微小胞の単離とキャラクタリゼーション

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

成人心臓線維芽細胞と筋線維芽細胞の分離と特徴付け

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

骨格筋線維芽細胞からのエクソソームの単離と特性評価

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

マウス初代肝臓正弦波内皮細胞の単離と特性評価

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...

Isolation and Characterization of Intact Phycobilisome in Cyanobacteria

In cyanobacteria, phycobilisome is a vital antenna protein complex that harvests light and transfers energy to photosystem I and II for photochemistry. Studying the structure and composition of phycobilisome is of great interest to scientists because it reveals the evolution and divergence of photosynthesis in cyanobacteria. This protocol provides a detailed and optimized method to break cyanobacterial cells at low cost by a bead-beater efficiently. The intact phycobilisome can then be isolated from the cell extract by sucrose gradient ultracentrifugation. This method has demonstrated being suitable for both model and non-model cyanobacteria with different cell types. A step-by-step procedure is also provided to confirm the integrity and property of phycobiliproteins by 77K fluorescence spectroscopy and SDS-PAGE stained by zinc sulfate and Coomassie Blue. The isolated phycobilisome can also be subjected to further structural and compositional analyses. Overall, this protocol provides a helpful starting guide that allows researchers unfamiliar with cyanobacteria to quickly isolate and characterize intact phycobilisome.

The current protocol details the isolation of phycobilisomes from cyanobacteria by centrifugation through a discontinuous sucrose density gradient. The fractions of intact phycobilisomes are confirmed by 77K fluorescent emission spectrum and SDS-PAGE analysis. The resulting phycobilisome fractions are suitable for negative staining of TEM and mass spectrometry analysis.

シアノバクテリアにおけるインタクトフィコビリソームの分離と特徴付け

In cyanobacteria, phycobilisome is a vital antenna protein complex that harvests light and transfers energy to photosystem I and II for photochemistry. ...

Isolation and Characterization of Cyanobacterial Extracellular Vesicles

Cyanobacteria are a diverse group of photosynthetic, Gram-negative bacteria that play critical roles in global ecosystems and serve as essential biotechnology models. Recent work has demonstrated that both marine and freshwater cyanobacteria produce extracellular vesicles -&#160;small membrane-bound structures released from the outer surface of the microbes. While vesicles likely contribute to diverse biological processes, their specific functional roles in cyanobacterial biology remain largely unknown. To encourage and advance research in this area, a detailed protocol is presented for isolating, concentrating, and purifying cyanobacterial extracellular vesicles. The current work discusses methodologies that have successfully isolated vesicles from large cultures of Prochlorococcus, Synechococcus, and Synechocystis. Methods for quantifying and characterizing vesicle samples from these strains are presented. Approaches for isolating vesicles from aquatic field samples are also described. Finally, typical challenges encountered with cyanobacterial vesicle purification, methodological considerations for different downstream applications, and the trade-offs between approaches are also discussed.

The present protocol providesdetailed descriptions for isolation, concentration,and characterization of extracellular vesicles from cyanobacterial cultures. Approaches for purifying vesicles from cultures at different scales, trade-offs among methodologies, and considerations for working with field samples are also discussed.

シアノバクテリア細胞外小胞の単離と特性評価

Cyanobacteria are a diverse group of photosynthetic, Gram-negative bacteria that play critical roles in global ecosystems and serve as essential ...

Isolation and Characterization of the Immune Cells from Micro-dissected Mouse Choroid Plexuses

The brain is no longer considered as an organ functioning in isolation; accumulating evidence suggests that changes in the peripheral immune system can indirectly shape brain function. At the interface between the brain and the systemic circulation, the choroid plexuses (CP), which constitute the blood-cerebrospinal fluid barrier, have been highlighted as a key site of periphery-to-brain communication. CP produce the cerebrospinal fluid, neurotrophic factors, and signaling molecules that can shape brain homeostasis. CP are also an active immunological niche. In contrast to the brain parenchyma, which is populated mainly by microglia under physiological conditions, the heterogeneity of CP immune cells recapitulates the diversity found in other peripheral organs. The CP immune cell diversity and activity change with aging, stress, and disease and modulate the activity of the CP epithelium, thereby indirectly shaping brain function. The goal of this protocol is to isolate murine CP and identify about 90% of the main immune subsets that populate them. This method is a tool to characterize CP immune cells and understand their function in orchestrating periphery-to-brain communication. The proposed protocol may help decipher how CP immune cells indirectly modulate brain function in health and across various disease conditions.

This study uses flow cytometry and two different gating strategies on isolated perfused mice brain choroid plexuses; this protocol identifies the main immune cell subsets that populate this brain structure.

微小解剖マウス脈絡叢からの免疫細胞の単離と特性評価

The brain is no longer considered as an organ functioning in isolation; accumulating evidence suggests that changes in the peripheral immune system can ...