この作品は、健康と嚢胞性線維症財団の国立研究所からの補助金によってサポートされていました。

1。 LPS抽出のための細菌の調製<ol><li>必要に応じて抗生物質を補充したルリア培地（LB）、5mlに一晩培養を開始します。 °C、200 rpmは37℃で振盪インキュベーターで一晩培養し（12-18時間）に成長。 </li><li> LBで培養1:10に希釈し、分光光度計でOD 600値を読み取ります。 OD 600が読みに基づいて、0.5のOD 600に細菌の1.5 mLの懸濁液を作る。 </li><li>ペレットを10分間10600×gで遠心中の細菌。上清を除去し、廃棄します。 LPSはすぐに抽出される予定されていない場合、ペレットは、-20℃で保存することができます。 </li></ol> 2。 LPSの抽出<ol><li>最初に、2倍のSDSバッファーを準備します。 4パーセントβ-メルカプトエタノール（BME）、0.1 Mトリス-HCl、pH 6.8で4％SDS、20％グリセロール50mLの溶液を作る。ソリューションを染めるためにブロモフェノールブルーのピンチを追加します。 2X sを希釈することにより、1×SDS-緩衝在庫を作る滅菌蒸留H 2 Oでタック午前1時01分これは室温で保存することができます。 </li><li>のDNase I、RNaseを、滅菌蒸留H 2 O中のプロテイナーゼKの3つの独立した10 mg / mLのソリューションを作成する</li><li> 1X SDS-緩衝液200μlのステップ1.3からペレット化細菌を再懸濁します。ペレットが完全にゆっくりと上下にソリューションをピペッティングにより再懸濁していることを確認します。ボルテックスしないでください。 </li><li> 15分間水浴中に懸濁させ、細菌を沸騰させる。ソリューションは、室温で15分間冷却することができます。 </li><li> 2.2で調製したDNase IとRNaseの両方のソリューションの5μlを追加します。 30分間37℃でサンプルをインキュベートします。 *このステップはオプションです。ヌクレアーゼ処理および非処理サンプル間のLPSの質の最小の差があります。 </li><li> 2.2で調製したプロティナーゼK溶液10μlを追加します。 3時間59°Cでサンプルをインキュベートします。時間の制約がある場合は、このステップは、一晩行うことができます。 </li><li>各サンプル、広告へのd 200μlの氷冷したトリス飽和フェノール（トリス飽和フェノールが利用できない場合は水飽和フェノールを代替として使用されるかもしれません）。チューブのキャップがしっかり閉じていることを確認し、約5〜10秒間ボルテックスし、各サンプル。 </li><li>時折ボルテックス、15分間65℃でサンプルをインキュベートします。室温まで冷却インキュベートした後、その後5〜10秒ごとにサンプルをボルテックスに室温でジエチルエーテル1mLを追加します。それは揮発性であるので、ドラフト内でハンドルエーテルを実行してください。 </li><li> 10分間、20600×gでサンプルを遠心分離します。遠心機からサンプルを削除して、底青色層を抽出します。上部の、明確な層を避けるようにしてください。青層に少量の後ろのままにしておくと、上位層の汚染に好適である。 </li><li> 2.9 - 手順2.7を繰り返して、サンプルを再抽出します。二つの抽出は、通常は十分です。サンプルは曇って表示される場合は、より多くの抽出は、performeかもしれませんD。 SDS-PAGEで分離する前に抽出したサンプルのそれぞれに2×SDS-緩衝液200μlを追加します。サンプルは、8％-15％SDS-ポリアクリルアミドゲル上で実行することができます。この方法を用いて調製したLPSの5〜15μlの可視化のために通常は十分です。 </li></ol> 3。代表的な結果上記のように調製したLPSのサンプルは、標準的な銀染色プロトコルまたは市販のLPS染色キットを用いたSDS-PAGE上で分離した後、直接染色により可視化することができます。また、ポリアクリルアミドゲル上で分離し、LPSをニトロセルロース膜に移し、LPS特異的抗血清を用いてイムノブロッティング西にさらされる可能性があります。このプロトコルのために、我々は、Pro-Qエメラルド300リポ多糖ゲル染色キット（Molecular Probes社）を使用し、製造元の指示に従った。 図1に示すようには、Pro-Qエメラルド300染色された12％SDS-ポリアクリルアミドゲルである。各レーンは、DIFから調製したLPSの15μlを含まれていますバークホルデリアdolosaのferent株は、嚢胞性線維症患者の喀痰検体から分離された。ラダーパターンをバンド別のLPSは、異なる数値型糖鎖コアに接続された繰り返し単位のO-抗原の反射は、このメソッドを使用して明白である。たとえば、レーン1と6のサンプルでは、​​お互いに類似したバンディングパターンを持っている（赤の囲み）。 <img alt="図1" src="/files/ftp_upload/3916/3916fig1.jpg" /> 図1プロQエメラルドバークホルデリアdolosa臨床分離株から300ステンドLPS。 7 BからのLPSこのプロトコルで説明するように嚢胞性線維症患者の発生からdolosa株が単離された。十五μlを12％SDS-ポリアクリルアミドゲル上で分離し、Pro-Qエメラルド300で染色し、製造元の指示に従って行った。 LPSコアは、矢印を示され、O抗原の繰り返し同様のバンドパターンを示したLPSは、赤で囲んだされています。 M =分子量マーカー。

<ol>
	<li>Raetz, C. R., Whitfield, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharide+endotoxins.">Lipopolysaccharide endotoxins.</a> Annu. Rev. Biochem. 71, 635-700 (2002).</li><li>Valvano, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Export+of+O-specific+lipopolysaccharide.">Export of O-specific lipopolysaccharide.</a> Front. Biosci. 8, 452-471 (2003).</li><li>Pier, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+lipopolysaccharide:+a+major+virulence+factor,+initiator+of+inflammation+and+target+for+effective+immunity.">Pseudomonas aeruginosa lipopolysaccharide: a major virulence factor, initiator of inflammation and target for effective immunity.</a> Int. J. Med. Microbiol. 297, 277-295 (2007).</li><li>Freudenberg, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+lipopolysaccharide+susceptibility+in+the+innate+immune+response+to+Salmonella+typhimurium+infection:+LPS,+a+primary+target+for+recognition+of+Gram-negative+bacteria.">Role of lipopolysaccharide susceptibility in the innate immune response to Salmonella typhimurium infection: LPS, a primary target for recognition of Gram-negative bacteria.</a> Microbes Infect. 3, 1213-1222 (2001).</li><li>Jann, K., Jann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysaccharide+antigens+of+Escherichia+coli.">Polysaccharide antigens of Escherichia coli.</a> Rev. Infect. Dis. 9, S517-S526 (1987).</li><li>McCallum, K. L., Schoenhals, G., Laakso, D., Clarke, B., Whitfield, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-molecular-weight+fraction+of+smooth+lipopolysaccharide+in+Klebsiella+serotype+O1:K20+contains+a+unique+O-antigen+epitope+and+determines+resistance+to+nonspecific+serum+killing.">A high-molecular-weight fraction of smooth lipopolysaccharide in Klebsiella serotype O1:K20 contains a unique O-antigen epitope and determines resistance to nonspecific serum killing.</a> Infect. Immun. 57, 3816-3822 (1989).</li><li>Hancock, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+isolates+from+patients+with+cystic+fibrosis:+a+class+of+serum-sensitive,+nontypable+strains+deficient+in+lipopolysaccharide+O+side+chains.">Pseudomonas aeruginosa isolates from patients with cystic fibrosis: a class of serum-sensitive, nontypable strains deficient in lipopolysaccharide O side chains.</a> Infect. Immun. 42, 170-177 (1983).</li><li>Gupta, S. K., Masinick, S., Garrett, M., Hazlett, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+lipopolysaccharide+binds+galectin-3+and+other+human+corneal+epithelial+proteins.">Pseudomonas aeruginosa lipopolysaccharide binds galectin-3 and other human corneal epithelial proteins.</a> Infect. Immun. 65, 2747-2753 (1997).</li><li>Hitchcock, P. J., Brown, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+heterogeneity+among+Salmonella+lipopolysaccharide+chemotypes+in+silver-stained+polyacrylamide+gels.">Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels.</a> J. Bacteriol. 154, 269-277 (1983).</li><li>Westphal, O., Jann, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+lipopolysaccharides.">Bacterial lipopolysaccharides.</a> Methods Carbohyr. Chem. 5, 83 (1965).</li><li>Lieberman, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+bacterial+evolution+within+multiple+patients+ties+novel+genes+to+pathogenesis.">Parallel bacterial evolution within multiple patients ties novel genes to pathogenesis.</a> Nat Genet. , (2011).</li></ol>

利害の衝突が宣言されません。

我々は、核酸やタンパク質を含む他の細胞成分から、LPSを離れて精製する方法を説明してきました。このメソッドは、図1に示すように、SDS-PAGEゲルの炭水化物染色など、さまざまな可視化手法、数で使用することができる高品質のLPSを提供しています。このメソッドは、特定の抗血清を使用して、株の様々なLPSの血清型に、または直接可視化による分離株の間に関連性を示すために使用する?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td> DNase Iを組換え、RNaseフリー</td><td>ロシュ社</td><td> 04716728001 </td></tr><tr><td> RNaseの</td><td>ロシュ社</td><td> 10109169001 </td></tr><tr><td>プロテイナーゼK </td><td>フィッシャー</td><td> BP1700 </td></tr><tr><td>フェノールトリス飽和</td><td>フィッシャー</td><td> BP1750-100 </td></tr><tr><td>ジエチルエーテル</td><td>トーマス科学</td><td> C313K31 </td></tr><tr><td>プロQエメラルド300リポ多糖ゲル染色キット</td><td>分子プローブ</td><td> P20495 </td></tr></tbody></table>

purification and visualization of lipopolysaccharide from gram-negative bacteria by hot aqueous-phenol extraction

リポポリサッカライド（LPS）はグラム陰性細菌の外膜の主要成分である。それは外側の膜に埋め込 ​​まれている、セル1、2の表面から外側に延びるコア糖鎖とO-抗原繰り返し単位脂質から成る三分子である。 LPSは、 緑膿菌、サルモネラ属、 大腸菌 3月5日を含む多くの細菌種の病原性と病原性に重要である免疫分子であり、LPS O抗原組成の違いは、株の血清型の基礎を形成する。 LPSは、感染の開始時に宿主細胞に付着に関与しており、補体媒介性の殺害からの保護を提供されています。LPSを欠いている株は病原性6-8減衰させることができる。これらの理由から、特に臨床分離株から、LPSを可視化することが重要です。具体的なパターンとの認識をバンディングの可視化LPSntibodiesは、ひずみの系統を識別するために、様々な変異体を特徴づけるために有用なツールにすることができます。 本稿では、グラム陰性細菌の細胞からのLPSの単離および精製のためのホット水 - フェノール法を説明します。このプロトコルは、距離が短く、より集中的な抽出方法9で発生したLPSの可視化を妨げる可能性核酸とタンパク質からLPSの抽出が可能になります。 LPSは、この方法は糖鎖/糖タンパク質汚れや標準の銀染色法を用いたドデシル硫酸ナトリウム（SDS） - ポリアクリルアミドゲル電気泳動（PAGE）と直接に染色されたで区切ることができます用意しました。 LPSには多くの抗血清では反応性を妨げることができる外膜タンパク質または他の抗原ターゲットと交差反応する抗体を含むSDS-PAGEで区切られた粗細胞溶解物のウエスタンブロット後に観察した。単独で粗細胞溶解物のプロテアーゼ処理は、常にこの除去の効果的な方法ではありませんこのまたは他の可視化方法を使用して背景。さらに、このような背景を削除しようとの広範なプロテアーゼ処理も、上述のいずれかの方法によって解決されていない低品質のLPSにつながる可能性があります。これらの理由から、我々はWestpahlとジャン10から適応し、次のプロトコルは、LPSの抽出のための理想的であると信じています。

Watch this Scientific Journal Video about Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction at JoVE.com

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

私たちは、グラム陰性菌からリポ多糖（LPS）を浄化するための変更されたホット水 - フェノール抽出法について説明します。一度抽出し、LPSは、その後、SDS-PAGEで分析し、直接染色またはウェスタンブロットによって可視化することができます。

ホット水 - フェノール抽出によるグラム陰性菌からの精製とリポ多糖の可視化

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

purification-visualization-lipopolysaccharide-from-gram-negative

Research

JoVE Journal

Immunology and Infection

35.0K ビュー.  University of Virginia Health System. 私たちは、グラム陰性菌からリポ多糖（LPS）を浄化するための変更されたホット水 - フェノール抽出法について説明します。一度抽出し、LPSは、その後、SDS-PAGEで分析し、直接染色またはウェスタンブロットによって可視化することができます。

ビデオ: Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

蛍光活性化細胞選別（FACS）によって特定の細胞集団の精製

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

免疫・感染学

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol (PEG) Precipitation

Hens can be immunized by means of i.m. vaccination (Musculus pectoralis, left and right, injection volume 0.5-1.0 ml) or by means of Gene-Gun plasmid-immunization. Dependent on the immunogenicity of the antigen, high antibody-titres (up to 1:100,000 - 1:1,000,000) can be achieved after only one or 3 - 4 boost immunizations. Normally, a hen lays eggs continuously for about 72 weeks, thereafter the laying capacity decreases. This protocol describes the extraction of total IgY from egg yolk by means of a precipitation procedure (PEG. Polson et al. 1980). The method involves two important steps. The first one is the removal of lipids and the second is the precipitation of total IgY from the supernatant of step one. After dialysis against a buffer (normally PBS) the IgY-extract can be stored at -20&deg;C for more than a year. The purity of the extract is around 80 %, the total IgY per egg varies from 40-80 mg, dependent on the age of the laying hen. The total IgY content increases with the age of the hen from around 40 mg/egg up to 80 mg/egg (concerning PEG precipitation). The laying capacity of a hen per year is around 325 eggs. That means a total potential harvest of 20 g total IgY/year based on a mean IgY content of 60 mg total IgY/egg (see Table 1).

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol PEG Precipitation

This protocol describes in particular the extraction of total IgY from egg yolk by means of polyethylene glycol precipitation and gives general information about IgY technology.

IGY技術：ポリエチレングリコール（PEG）沈殿により卵黄からニワトリ抗体の抽出

Hens can be immunized by means of  i.m. vaccination (Musculus pectoralis, left and right, injection volume  0.5-1.0 ml) or by means of Gene-Gun ...

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

からのDNA抽出のためのシンプルなChelexプロトコル<em&gt;ハマダラカ</em&gt;属。

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe pneumonia "Legionnaires' disease"1. In protozoan and mammalian phagocytes, L. pneumophila employs a conserved mechanism to form a specific, replication-permissive compartment, the "Legionella-containing vacuole" (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS), which translocates as many as 275 "effector" proteins into host cells. The effectors manipulate host proteins as well as lipids and communicate with secretory, endosomal and mitochondrial organelles2-4.
The formation of LCVs represents a complex, robust and redundant process, which is difficult to grasp in a reductionist manner. An integrative approach is required to comprehensively understand LCV formation, including a global analysis of pathogen-host factor interactions and their temporal and spatial dynamics. As a first step towards this goal, intact LCVs are purified and analyzed by proteomics and lipidomics.
The composition and formation of pathogen-containing vacuoles has been investigated by proteomic analysis using liquid chromatography or 2-D gel electrophoresis coupled to mass-spectrometry. Vacuoles isolated from either the social soil amoeba Dictyostelium discoideum or mammalian phagocytes harboured Leishmania5, Listeria6, Mycobacterium7, Rhodococcus8, Salmonella9 or Legionella spp.10. However, the purification protocols employed in these studies are time-consuming and tedious, as they require e.g. electron microscopy to analyse LCV morphology, integrity and purity. Additionally, these protocols do not exploit specific features of the pathogen vacuole for enrichment.
The method presented here overcomes these limitations by employing D. discoideum producing a fluorescent LCV marker and by targeting the bacterial effector protein SidC, which selectively anchors to the LCV membrane by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P)3,11 . LCVs are enriched in a first step by immuno-magnetic separation using an affinity-purified primary antibody against SidC and a secondary antibody coupled to magnetic beads, followed in a second step by a classical Histodenz density gradient centrifugation12,13 (Fig. 1).
A proteome study of isolated LCVs from D. discoideum revealed more than 560 host cell proteins, including proteins associated with phagocytic vesicles, mitochondria, ER and Golgi, as well as several GTPases, which have not been implicated in LCV formation before13. LCVs enriched and purified with the protocol outlined here can be further analyzed by microscopy (immunofluorescence, electron microscopy), biochemical methods (Western blot) and proteomic or lipidomic approaches.

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

This article describes a method for the isolation and purification of intact Legionella-containing vacuoles (LCVs) from amoeba and macrophages. The two-step protocol comprises LCV enrichment by immuno-magnetic separation using an antibody against a bacterial LCV marker and further purification by density gradient centrifugation.

病原体胞の精製から<em&gt;レジオネラ菌</em&gt;感染した食細胞

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

MALDI-TOF質量分析法を使用した血液培養ブロスからのグラム陰性菌の迅速な同定

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

T細胞は、樹状細胞からTransinfectionによって細菌をキャプチャ

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

RNA精製は、細胞内から成長します<em&gt;リステリア菌</em&gt;マクロファージ細胞で

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with respect to how viral members of the microbiome affect subsequent microbe interactions with these virus-infected host cells. The co-habitation of virus with bacteria and fungi is principally present on the mucosal surfaces of the oral cavity and genital tract. Mucosal cells, particularly those with persistent chronic or persistent latent viral infections, could have a significant impact on members of the microbiome through virus alteration in number and type of receptors expressed. Modification in host cell membrane architecture would result in altered ability of subsequent members of the normal flora and opportunistic pathogens to initiate the first step in biofilm formation, i.e., adherence. This study describes a method for quantitation and visual examination of HSV&#39;s effect on the initiation of biofilm formation (adherence) of S. aureus and C. albicans.

A method is described herein for the determination of inter-Kingdom association and competition (bacterial and fungal) for adherence to virus-infected HeLa cell monolayers. This protocol can be extended to multiple combinations of prokaryotes, eukaryotes, and viruses.

細菌や真菌によってウイルス感染細胞上のバイオフィルムの開始の決意

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

肥満に関連する炎症によって影響を受ける組織のマクロファージの精製と性質、分離

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 &#215; 106 live hemocytes can be obtained from 200 Drosophila 3rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

This method demonstrates how to visualize pathogen invasion into insect cells with three-dimensional (3D) models. Hemocytes from Drosophila larvae were infected with viral or bacterial pathogens, either ex vivo or in vivo. Infected hemocytes were then fixed and stained for imaging with a confocal microscope and subsequent 3D cellular reconstruction.

細菌感染や解析のためのキイロショウジョウバエ幼虫血球の抽出

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...

ホット水 - フェノール抽出によるグラム陰性菌からの精製とリポ多糖の可視化

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