Name: isolation and characterization of extracellular vesicles from adult schistosoma japonicum
Uploaded: 2018-05-22T13:00:00.000+00:00
Duration: 7 min 37 s
Description: Watch this Scientific Journal Video about Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum at JoVE.com

本研究は、一部または全部、国家自然科学基金、中国の (31472187 および 31672550) および農業科学と中国農業科学院の技術革新プログラムでサポートされています。

上海中国農業科学院獣医研究所倫理委員会で承認されたすべての動物実験 (許可番号: SHVRIAU-14-0101)。
1住の体外培養。
注:住は、バイオハザードを表しています。労働者は関連生物学的材料をワーム、感染後懸濁液、または他を処理する場合もすべてのラテックス手袋を着用ください。ニュージーランドのウサギは、腹部の被曝経由~ 2,000 セルカリアに感染していた。肝の段階で住は肝臓と腸間膜静脈の血流で 28 日後の感染症で米住血吸虫セルカリアに感染ウサギから収集されました。ワーム コレクションのプロシージャは、マウス18研究報告以前の出版物で参照されています。
<ol><li>500 mL ビーカーに寄生虫を収集します。それらを徹底的に洗浄し、200 ml の軽く 3 倍予熱 (37 ° C) PBS (pH 7.4)。</li>
	<li>各 90 mm のシャーレの ~ 200 ワームのペアを区切ります。寄生虫を徹底的に洗うし、軽く 2 倍の 50 mL の予熱 (37 ° C) RPMI 1640 培養液中のペニシリンとストレプトマイシンの 100 mg/mL 100 U。</li>
	<li>37 ° C 未満 5 %90 mm ペトリで h 2 〜 5 ワームのペア/mL の密度で CO2皿で抗生物質なし予熱した RPMI 1640 培養液 40 mL に寄生虫を維持します。 ノート:RPMI 1640 培地に血清を含めることはできません、それ以外の場合、外因性細胞小胞が導入されます。</li>
</ol>2. 前処理条件メディア
<ol><li>培養液を収集し、2,000 × g の卵を削除し、被蓋の破片をワーム 30 分で 4 ° C にある媒体を遠心分離します。</li>
	<li>新鮮なチューブに上清を転送し、0.22 μ m シリンジ フィルターを使用して、ソリューションをフィルターします。</li>
	<li>透析バッグ フィルタ リング ソリューションを収集 (分子量カットオフ: 3.5kD)、一晩 4 ° C で 8 %peg8000 溶液を使用して、ソリューションを dialyze と。 ノート:この手順は、SjEVs 分離の収量を高めるため SjEVs を豊かにすることが重要です。</li>
</ol>3. SjEVs の単離
<ol><li>透析のソリューションを新しいチューブに転送し、0.5 合計エキソソーム分離試薬量を追加します。</li>
	<li>ボルテックスやソリューションの均一性を確保するため、上下ピペッティングでよくソリューションをミックスします。</li>
	<li>2 の ° C ~ 8 ° C で混合物を一晩インキュベートします。</li>
	<li>2 の ° C ~ 8 ° C で 1 時間 15,000 × g で混合物を遠心分離します。</li>
	<li>吸引し、上澄みを廃棄します。 ノート:EVs は、(ほとんどの場合に表示されません) の管の下部にペレットに含まれています。</li>
	<li>2 mL の PBS で EVs ペレットを再懸濁し、詳細な分析まで-80 ° C で EVs ソリューションします。</li>
</ol>4 西部にしみが付くことによって Ev の特性
<ol><li>例えば、 BCA アッセイで SjEVs サンプルのタンパク質濃度を決定します。</li>
	<li>22.5 μ L RIPA バッファー内の EVs の 10-20 μ g を準備します。バッファーおよび 95 ° C で 5 分間熱ロード x 4 の 7.5 μ L を追加します。</li>
	<li>SjEVs タンパク質を 12 %sds ページのゲルに区切ります。</li>
	<li>PVDF 膜に蛋白質を転送および抗うさぎ IgG HRP (1:5, 000 希釈) に調査続く反 CD63 抗体 (1:1, 000 の希釈) をプローブします。</li>
	<li>膜開発の ECL 検出試薬を使用し、化学発光イメージング システムによって信号を検出します。</li>
</ol>5. 電子顕微鏡による Ev の特性
<ol><li>200 メッシュ ホルムバール コーティング グリッドに孤立した SjEVs の 2 μ l 添加し、室温で乾燥すること。</li>
	<li>1 分リンタングステン酸 2% で染色し、室温で乾燥します。</li>
	<li>透過型電子顕微鏡の試料ホルダーの上にグリッドをロードし、イメージのキャプチャ用 80 kV 電子ビームにさらされています。</li>
</ol>6. SjEVs ラベル PKH67 と細胞取り込みアッセイ
ノート:これ以降の手順は、特に明記しない限り周囲温度 (20-25 ° C) で行われました。
<ol><li>円錐底部ポリプロピレン チューブに 5 μ g SjEVs ソリューションを転送し、RPMI 1640 媒体を追加して 12 mL にボリュームを調整します。</li>
	<li>SjEVs のペレットへの 4 ° C で 90 分 110,000 × g で SjEVs を遠心します。</li>
	<li>遠心分離の後上澄みを慎重に吸引します。 注:これは異種染色および減らされた細胞生存率になると、SjEV ペレットに直接 PKH67 アルコール染料を追加しないでください。</li>
	<li>SjEV ペレット; に 1 mL の希釈剤 C を追加して 2 x EV 停止ソリューションを準備します。完全な分散するように、穏やかなピペッティングで再懸濁します。</li>
	<li>希釈ハ染色直前に希釈 C の 1 ml ポリプロピレン製遠心管に PKH67 エタノール色素溶液 2 μ L を追加し、分散させるためによく混合色素溶液 x 新鮮な 2 を準備します。</li>
	<li>EV 停止 (ステップ 6.4) 2 x の染料の解決 (手順 6.5) とすぐに 1 mL にピペットでサンプルをミックス 2 x の 1 mL を急速に追加します。</li>
	<li>4 分間室温で混合物を孵化させなさい。</li>
	<li>FBS の 4 つの mL を追加することによって染色を停止し、1 分間インキュベートします。</li>
	<li>12 mL にボリュームを調整して、90 分の 4 ° C で 110,000 × g で遠心分離 RPMI1640 培地を追加します。</li>
	<li>上澄みを除去し、PKH67 ラベル SjEV ペレットを再懸濁しますに RPMI1640 培地を追加します。</li>
	<li>90 分 1 回洗浄し、SjEVs ペレットを再懸濁しますに PBS を追加するための 4 ° C で 110,000 × g で遠心分離機します。</li>
</ol>7. SjEVs 細胞取り込みアッセイ
<ol><li>NCTC など種の哺乳類細胞 1469 セルまたは 6 ウェル プレート (2 × 105細胞/ウェル) HEK293T 細胞を複製し、一晩培養します。</li>
	<li>5 μ g PKH67 ラベル SjEVs を追加し、37 ° C 2-4 h での細胞の培養します。</li>
	<li>インキュベーション後、メディアを取り出して、PBS のセルを 2 回洗います。</li>
	<li>4% ホルムアルデヒド溶液中 15 分間とセルを修正し、PBS で 2 回洗浄します。</li>
	<li>4 ', 6-diamidino-2-phenylindole (DAPI) と細胞核を染色します。</li>
	<li>蛍光顕微鏡を用いた細胞を観察します。</li>
</ol>

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Ev に関する最近の研究は、EVs は、宿主-病原体相互作用3,9,12,16で重要な役割を果たす、住血吸虫を実証しています。さらにアドレスに規制機能のそれは住から EVs を分離するため不可欠であります。ここでは、SjEV の分離のための代替方法をについて説明します。このメソッドは、100 から、SjEVs の...

細胞小胞 (Ev) は、様々 なタンパク質、脂質、核酸でカプセル化されます小さな膜結合型ベシクルの人口です。EVs が細胞間の情報伝達に重要な役割を果たして、細胞の分化、免疫調節、血管新生、セル移行2など多数の生理学的なプロセスの規則にかかわる最近の研究を実証 3,4,5。証拠の蓄積は、EVs を, 循環エクソソーム、および miRNA 貨物が特定疾患6の潜在的なバイオ マーカーを表すことを示します。
トリコモナス、 cruzi トリパノソーマの原因、リーシュ マニア属などのいくつかの原生動物、EVs; を分泌することができることが示されている。寄生虫が見つかってまた7をホストの生活に EVs を分泌します。寄生 EVs 感染、病原性8、および免疫調節9の維持に関与することが示されています。住での最近の調査は両方のマンソン住血吸虫(マンソン住血吸虫など) 10、11 S. ササユリ12アダルト住がありますエキソソームのような小胞を分泌することが示されています。特定の生物学的プロセスの機能の規則にかかわる。
日には、超遠心法、限外濾過法13イムノアフィニティーカラム分離、サイズ排除クロマトグラフィー, ポリマー ベースの試薬の使用など、細胞の小胞を分離するいくつかの方法を使用されています。これらの異なる方法では、独自の利点と制限14を所有しています。一般的に、遠心は小胞の分離のためのゴールド スタンダード メソッドと見なされます。ただし、このメソッドは、潜在的な EV 集計14の制限に苦しみます。
住、いくつかのメソッドは、EV の分離報告されている: 遠心11,12,10といくつかの段階 (卵16、商用 EV 分離キット16の利用が含まれますschistosomula11, アダルト住10,12,17)。ベンチの遠心分離機、限外濾過から EVs を分離するための市販 EV 分離キットの使用を組み合わせた代替法を開発した機構の解明は、1000 ナノメートル数百ナノメートルからのサイズの広い範囲が、アダルト日本住血吸虫。分離の EVs は、通常 100-400 nm の直径を所有し、正常に受信者の細胞によって内面化されました。

<table><tbody><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Total Exosome Isolation Reagent (from cell culture media)</td><td>ThermoFisher SCIENTIFIC</td><td>4478359</td><td>For isolating S. japonicum extracellular vesicles</td></tr><tr><td>PKH67</td><td>Sigma-aldrich</td><td>MINI67</td><td>For labeling Evs</td></tr><tr><td>RPMI 1640 Medium</td><td>ThermoFisher SCIENTIFIC</td><td>11875119</td><td>For parasite culture</td></tr><tr><td>Penicillin-Streptomycin</td><td>ThermoFisher SCIENTIFIC</td><td>15140122</td><td></td></tr><tr><td>0.22&amp;mu;m syringe filter</td><td>Merck</td><td>SLGP033RB</td><td></td></tr><tr><td>SnakeSkin Dialysis Tubing</td><td>Thermo SCIENTIFIC</td><td>68035</td><td></td></tr><tr><td>PEG8000</td><td>Sangon Biotech</td><td>A100159&amp;nbsp;</td><td></td></tr><tr><td>RIPA</td><td>Beyotime</td><td>P0013B</td><td></td></tr><tr><td>EMD Millipore&amp;trade; Immobilon&amp;trade; Western Chemiluminescent HRP Substrate (ECL)</td><td>Fisher scientific</td><td>WBKLS0100&amp;nbsp;</td><td></td></tr><tr><td>DAPI</td><td>Cell Signaling Technology</td><td>4083</td><td></td></tr><tr><td>BCA kit</td><td>Beyotime</td><td>P0010</td><td></td></tr><tr><td>CD63 antibodies</td><td>Sangon Biotech</td><td>D160973-0025</td><td></td></tr><tr><td>PVDF</td><td>Bio-Rad</td><td>1704273</td><td></td></tr><tr><td>Formvar/Carbon 400 mesh</td><td>Ted Pella</td><td>01754-F</td><td></td></tr><tr><td>Phosphotungstic Acid</td><td>Ted Pella</td><td>19402</td><td></td></tr><tr><td>anti-mouse IgG-HRP</td><td>CWBIO</td><td>CW0102</td><td></td></tr><tr><td>NCTC clone 1469 cells</td><td>ATCC</td><td>ATCC&amp;reg; CCL-9.1&amp;trade;</td><td></td></tr><tr><td>FBS</td><td>HyClone</td><td>SV30087.02</td><td></td></tr><tr><td>&lt;strong&gt;Equipments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>GE chemoluminescance imaging system</td><td>GE</td><td>ImageQuant LAS4000mini</td><td></td></tr><tr><td>Transmission electron microscopy</td><td>Hitachi</td><td>H-7600</td><td></td></tr><tr><td>Milli-Q water</td><td>Milli-Q</td><td>Milli-Q Elix</td><td></td></tr><tr><td>Eppendor Centrifuge&amp;nbsp;</td><td>Eppendorf</td><td>Centrifuge 5804R</td><td></td></tr><tr><td>Zetasizer Nano</td><td>Malvern</td><td>Zetasizer Nano ZS&amp;nbsp;</td><td></td></tr><tr><td>Ultracentrifuge</td><td>Beckman</td><td>Optima L-100 XP Ultracentrifuge</td><td></td></tr><tr><td>Incubator</td><td>ESCO</td><td>CCL-107B</td><td></td></tr><tr><td>Microscope</td><td>Zeiss</td><td>Zeiss Axin Observer Z1</td><td></td></tr></tbody></table>

isolation and characterization of extracellular vesicles from adult schistosoma japonicum

細胞小胞 (Ev) は、膜小胞の細胞外微小環境に様々 な細胞によって放出です。EVs は、異種の小胞の人口は 40 と 1,000 nm 間のサイズの範囲を表しています。蓄積された証拠は、EVs がホスト病原体の相互作用で重要な規制の役割を果たすことを示した。住血吸虫の EVs の深い理解は、住血吸虫ホスト相互作用、住血吸虫症に対する新しい戦略の開発を有効にするメカニズムに洞察力を提供しなければなりません。ここでは、さらにアダルト日本住血吸虫(S. ササユリ) から分離・同定 EVs のためのプロトコルを提示による住で EVs 機能を研究を目指しています。EVs は体外培養液遠心商業エキソソーム分離キットと組み合わせての使用から分離しました。分離されたs. ササユリEVs (SjEVs) は通常 100-400 nm の直径を持つし、透過電子顕微鏡と西部にしみが付くことによって特徴付けられます。PKH67 色素標識 SjEVs の使用は、SjEVs は受信者の細胞によって内面化を実証しています。全体として、我々 のプロトコルは大人住; から EVs を分離するための代替方法を提供します。分離 SjEVs の機能解析が適さない場合があります。

記述のプロトコルを使用して孤立した SjEVs の収量を定量化するには、28 日アダルト住から分離された SjEVs の蛋白質の集中をアクセスするのに BCA タンパク質アッセイを使用しました。表 1に示すように、SjEV 蛋白質の集中は媒体 (表 1) の 100 mL あたり 250 μ g 208 μ g からであった。粒子サイズ モールヴァン ナノ粒子解析によって決定される表?...

Watch this Scientific Journal Video about Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum at JoVE.com

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

分離と大人住血吸虫から細胞外小胞のキャラクタリゼーション

ここでは、アダルト住血吸虫から培養体外細胞小胞 (Ev) の分離を記述するプロトコルを提案する.

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...

isolation-characterization-extracellular-vesicles-from-adult

Research

JoVE Journal

Immunology and Infection

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

7.8K ビュー.  Ministry of Agriculture. ここでは、アダルト住血吸虫から培養体外細胞小胞 (Ev) の分離を記述するプロトコルを提案する.

ビデオ: Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ranks second to malaria among parasitic diseases in terms of public health and socio-economic impact (1-4). Schistosome parasites are trematode worms with a complex life cycle interchanging between a parasitic life in molluscan and mammalian hosts with intervening free-swimming stages. Briefly, free-swimming cercariae infect a mammalian host by penetrating the skin with the aid of secreted proteases, during which time the cercariae lose their tails, transforming into schistosomules. The schistosomules must now evade the host immune system, develop a gut for digestion of red blood cells, and migrate though the lungs and portal circulation en route to their final destination in the hepatic portal system and eventually the mesenteric veins (for S. mansoni) where male and female worms pair and mate, producing hundreds of eggs daily. Some of the eggs are excreted from the body into fresh water, where the eggs hatch into free-swimming miracidia (5-10). The miracidia infect specific snail species and transform into mother and daughter sporocysts, which in turn, produce infective cercariae, completing the life cycle. Unfortunately, the entire schistosome life cycle cannot be cultured in vitro, but infective cercariae can be transformed into schistosomules, and the schistosomules can be cultured for weeks for the analysis of schistosome development in vitro or microarray analysis. In this protocol, we provide a visual description of cercarial transformation and in vitro culturing of schistosomules. We shed infectious cercariae from the snail host Biomphalaria glabrata and manually transform them into schistosomules by detaching their tails using an emulsifying double-ended needle. The in vitro cercarial transformation and schistosomules culture techniques described avoid the use of a mammalian host, which simplifies visualization of schistosomes and facilitates the collection of the parasite for experimental analysis. in vitro transformation and culturing techniques of schistosomes have been done for years (11, 12), but no visual protocols have been developed that are available to the entire community.

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

We describe an in vitro method for culturing schistosomula of the flatworm parasite Schistosoma mansoni, via the harvesting and transformation of infective cercariae from the fresh water snail intermediate host Biomphalaria glabrata.

セルカリア変換と in vitroで</emの>栽培マンソン住血吸虫 Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ...

免疫・感染学

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe pathology, including liver fibrosis and splenomegaly, is caused by the immune response to the parasite eggs rather than the parasite itself 2. Parasite eggs induce a Th2 response characterized by the production of IL-4, IL-5 and IL-13, the alternative activation of macrophages and the recruitment of eosinophils. Here, we describe injection of Schistosoma mansoni eggs as a model to examine parasite-specific Th2 cytokine responses in the lung and draining lymph nodes, the formation of pulmonary granulomas surrounding the egg, and airway inflammation. 
Following intraperitoneal sensitization and intravenous challenge, S. mansoni eggs are transported to the lung via the pulmonary arteries where they are trapped within the lung parenchyma by granulomas composed of lymphocytes, eosinophils and alternatively activated macrophages 3-6. Associated with granuloma formation, inflammation in the broncho-alveolar spaces, expansion of the draining lymph nodes and CD4 T cell activation can be observed. Here we detail the protocol for isolating Schistosoma mansoni eggs from infected livers (modified from 7), sensitizing and challenging mice, and recovering the organs (broncho-alveolar lavage (BAL), lung and draining lymph nodes) for analysis. We also include representative histologic and immunologic data and suggestions for additional immunologic analysis. 
Overall, this method provides an in vivo model to investigate helminth-induced immunologic responses in the lung, which is broadly applicable to the study of Th2 inflammatory diseases including helminth infection, fibrotic diseases, allergic inflammation and asthma. Advantages of this model for the study of type 2 inflammation in the lung include the reproducibility of a potent Th2 inflammatory response in the lung and draining lymph nodes, the ease of assessment of inflammation by histologic examination of the granulomas surrounding the egg, and the potential for long-term storage of the parasite eggs.

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.

から卵を使用して、<em&gt;マンソン住血吸虫</em&gt;として<emインビボ&gt;</em蠕虫によって誘発される肺の炎症の&gt;モデル

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe ...

Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni

Human blood flukes, Schistosoma spp., have a complex life cycle that involves asexual and sexual developmental phases within a snail intermediate and mammalian final host, respectively. The ability to isolate and sustain the different life cycle stages under in vitro culture conditions has greatly facilitated investigations of the cellular, biochemical and molecular mechanisms regulating parasite growth, development and host interactions. Transmission of schistosomiasis requires asexual reproduction and development of multiple larval stages within the snail host; from the infective miracidium, through primary and secondary sporocysts, to the final cercarial stage that is infective to humans. In this paper we present a step-by-step protocol for mass hatching and isolation of Schistosoma mansoni miracidia from eggs obtained from livers of infected mice, and their subsequent introduction into in vitro culture. It is anticipated that the detailed protocol will encourage new researchers to engage in and broaden this important field of schistosome research.

Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni

This article describes a protocol for the large-scale axenic isolation and collection of free-swimming miracidia of the human blood fluke Schistosoma mansoni and their subsequent processing for introduction into in vitro culture.

質量分離と人間住血吸虫マンソン住血吸虫の Intramolluscan の段階の体外培養

Human blood flukes, Schistosoma spp., have a complex life cycle that involves asexual and sexual developmental phases within a snail intermediate and ...

発生生物学

An Enrichment Method for Small Extracellular Vesicles Derived from Liver Cancer Tissue

Small extracellular vesicles (sEVs) derived from tissue can reflect the functional status of the source cells and the characteristics of the tissue's interstitial space. The efficient enrichment of these sEVs is an important prerequisite to the study of their biological function and a key to the development of clinical detection techniques and therapeutic carrier technology. It is difficult to isolate sEVs from tissue because they are usually heavily contaminated. This study provides a method for the rapid enrichment of high-quality sEVs from liver cancer tissue. The method involves a four-step process: the incubation of digestive enzymes (collagenase D and DNase &#921;) with tissue, filtration through a 70 &#181;m cell strainer, differential ultracentrifugation, and filtration through a 0.22 &#181;m membrane filter. Owing to the optimization of the differential ultracentrifugation step and the addition of a filtration step, the purity of the sEVs obtained by this method is higher than that achieved by classic differential ultracentrifugation. It provides an important methodology and supporting data for the study of tissue-derived sEVs.

Here we describe the enrichment of small extracellular vesicles derived from liver cancer tissue through an optimized differential ultracentrifugation method.

肝癌組織由来の微小細胞外小胞の濃縮法

Small extracellular vesicles (sEVs) derived from tissue can reflect the functional status of the source cells and the characteristics of the tissue's ...

がん研究

Size Exclusion Chromatography for Separating Extracellular Vesicles from Conditioned Cell Culture Media

Extracellular vesicles (EVs) are nano-sized lipid-membrane bound structures that are released from all cells, are present in all biofluids, and contain proteins, nucleic acids, and lipids that are reflective of the parent cell from which they are derived. Proper separation of EVs from other components in a sample allows for characterization of their associated cargo and lends insight into their potential as intercellular communicators and non-invasive biomarkers for numerous diseases. In the current study, oligodendrocyte derived EVs were isolated from cell culture media using a combination of state-of-the-art techniques, including ultrafiltration and size exclusion chromatography (SEC) to separate EVs from other extracellular proteins and protein complexes. Using commercially available SEC columns, EVs were separated from extracellular proteins released from human oligodendroglioma cells under both control and endoplasmic reticulum (ER) stress conditions. The canonical EV markers CD9, CD63, and CD81 were observed in fractions 1-4, but not in fractions 5-8. GM130, a protein of the Golgi apparatus, and calnexin, an integral protein of the ER, were used as negative EV markers, and were not observed in any fraction. Further, when pooling and concentrating fractions 1-4 as the EV fraction, and fractions 5-8 as the protein fraction, expression of CD63, CD81, and CD9 in the EV fraction was observed. The expression of GM130 or calnexin was not observed in either of the fraction types. The pooled fractions from both control and ER stress conditions were visualized with transmission electron microscopy and vesicles were observed in the EV fractions, but not in the protein fractions. Particles in the EV and protein fractions from both conditions were also quantified with nanoparticle tracking analysis. Together, these data demonstrate that SEC is an effective method for separating EVs from conditioned cell culture media.

The protocol here demonstrates that extracellular vesicles can be adequately separated from conditioned cell culture media using size exclusion chromatography.

馴化細胞培養培地から細胞外小胞を分離するためのサイズ排除クロマトグラフィー(英語)

Extracellular vesicles (EVs) are nano-sized lipid-membrane bound structures that are released from all cells, are present in all biofluids, and contain ...

神経科学

Isolation And Dendritic Cell-Uptake of Small Extracellular Vesicles from Echinococcus granulosus

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In particular, small extracellular vesicles (sEVs) act as nano-carriers transferring natural antigens, which are critical in host immunomodulation and parasite survival. This article presents a step-by-step protocol to isolate sEVs from larval stage cultures of Echinococcus granulosus and analyzes their uptake by dendritic cells obtained from murine bone marrow, which acquire adhesion and antigen presentation capacity during their maturation after one week of in vitro culture. This article provides comprehensive information for generating, purifying, and quantifying sEVs using ultracentrifugation alongside parallel analyses of dynamic light scattering and transmission electron microscopy. Additionally, a detailed experimental protocol is outlined for isolating and cultivating mouse bone marrow cells and driving their differentiation into dendritic cells using Flt3L. These dendritic cells can present antigens to na&#239;ve T cells, thereby modulating the type of immune response in vivo. Thus, alternative protocols, including confocal microscopy and flow cytometry analysis, are proposed to check the acquired maturational phenotype of dendritic cells previously exposed to parasitic sEVs. Finally, it is worth noting that the described protocol can be applied as a whole or in individual parts to carry out parasite in vitro culture, isolate extracellular vesicles, generate bone marrow-derived dendritic cell cultures, and perform uptake assays with these cells.

Here, we describe in vitro culture conditions, isolation, and increased generation of extracellular vesicles (EVs) from Echinococcus granulosus. The small EVs were characterized by dynamic light scattering and transmission electron microscopy. The uptake by bone marrow-derived dendritic cells and their phenotypic modulation were studied using confocal microscopy and flow cytometry.

Echinococcus granulosusからの小さな細胞外小胞の単離と樹状細胞の取り込み

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

細胞外小胞体のカエノールハブディティス・エレガンスの精製と分析

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

生物学

<meta charset="utf8"></head><body>マウス脾臓由来エクソソーム単離のための精製技術

Isolation and Characterization of Exosomes Derived from Mouse Spleen Tissues

Exosomes (Exo) are lipid-bilayer structures secreted by various cells, including those of animals, plants, and prokaryotes. Previous studies have revealed that Exo derived from humoral or cell-supernatant are promising targets for novel diagnostic or prognostic biomarkers, underscoring their significant role in disease pathogenesis. Tissue-derived Exo (Ti-Exo) have attracted increasing attention due to its ability to accurately reflect tissue specificity and the microenvironment. Ti-Exo, present in interstitial space, play crucial roles in intercellular communication and cross-organ signaling. Despite their recognized value in elucidating disease mechanisms, isolating Ti-Exo remains challenging due to the complexity of tissue matrices and variability in extraction methods. In this study, we developed a practical protocol for isolating exosomes from mice spleen tissue, providing a reproducible technique for subsequent identification analysis and functional studies. We used Type I collagenase digestion combined with differential ultracentrifugation to isolate spleen-derived Exo. The characteristics of isolated Exo were determined through electron microscopy, the nano-flow cytometer, and the western blot. The isolated spleen-derived Exo displayed the typical morphology of lipid bilayer vesicles, with particle sizes ranging from 30 nm to 150 nm. In addition, the expression profile of exosome markers confirmed the presence and purity of exosomes. Taken together, we successfully established a practical protocol for isolating spleen-derived Exo in mice.

<meta charset="utf8"></head><body>マウス脾臓組織由来エクソソームの単離と特性評価

Here, we present a protocol to isolate mouse spleen-derived exosomes using a combination of digestion with collagenase type I and ultracentrifugation.

マウス脾臓組織由来エクソソームの単離と特性評価

Exosomes (Exo) are lipid-bilayer structures secreted by various cells, including those of animals, plants, and prokaryotes. Previous studies have revealed ...

分離と大人住血吸虫から細胞外小胞のキャラクタリゼーション

要約

さらに動画を探す

この動画の章

セルカリア変換と in vitroで栽培マンソン住血吸虫* Schistosomules*

から卵を使用して、

質量分離と人間住血吸虫マンソン住血吸虫の Intramolluscan の段階の体外培養

肝癌組織由来の微小細胞外小胞の濃縮法

馴化細胞培養培地から細胞外小胞を分離するためのサイズ排除クロマトグラフィー(英語)

Echinococcus granulosusからの小さな細胞外小胞の単離と樹状細胞の取り込み

細胞外小胞体のカエノールハブディティス・エレガンスの精製と分析

マウス脾臓組織由来エクソソームの単離と特性評価

マウス脾臓組織由来エクソソームの単離と特性評価

マウス脾臓組織由来エクソソームの単離と特性評価

分離と大人住血吸虫から細胞外小胞のキャラクタリゼーション

要約

さらに動画を探す

この動画の章

セルカリア変換と in vitroで栽培マンソン住血吸虫 Schistosomules

から卵を使用して、

質量分離と人間住血吸虫マンソン住血吸虫の Intramolluscan の段階の体外培養

肝癌組織由来の微小細胞外小胞の濃縮法

馴化細胞培養培地から細胞外小胞を分離するためのサイズ排除クロマトグラフィー(英語)

Echinococcus granulosusからの小さな細胞外小胞の単離と樹状細胞の取り込み

細胞外小胞体のカエノールハブディティス・エレガンスの精製と分析

マウス脾臓組織由来エクソソームの単離と特性評価

マウス脾臓組織由来エクソソームの単離と特性評価

マウス脾臓組織由来エクソソームの単離と特性評価

セルカリア変換と in vitroで栽培マンソン住血吸虫* Schistosomules*