著者は、ビデオ録画や編集の Ander ロペスとみれん Telletxea の支援を認めます。ACR は、Ikerbasque ・ フェローシップ ・隣人・ デ ・ Celiacos マドリード (ACM) から研究プロジェクトによって資金を供給します。JRB はプロジェクト ISCIII-PI16/00258 によって資金を供給、共同欧州連合 ERDF/ESF「ヨーロッパをさせる方法」によって資金を供給されます。バスク保健省の研究プロジェクト助成 2015111068 によって資金を供給されます。IRG と AJM それぞれ UPV/EHU とバスク語教育部門からまた、博士課程フェローシップ助成金によってサポートされます。

本研究はラスクルーセス大学病院倫理委員会によって承認された、すべての科目や両親からインフォームド コンセントを得た後分析を行った。
1. 生検コレクション
注: 患者の遠位十二指腸生検重量 2 〜 10 mg 間の範囲の通常の診断内視鏡生検の管理中に取得されます。生検組織は、異なる細胞など上皮、免疫および血管内皮細胞から成る複雑な組織です。臨床消化器内科内視鏡検査を実行する確立された臨床ガイドラインに従う。
<ol><li>内視鏡検査後だけ滅菌フィルター ペーパーに生検を配置します。</li>
	<li>1.5 mL チューブに生検を転送し、PBS でそれらを浸し、cryotube に鉗子を使用します。</li>
	<li>研究室に到着するまで、氷の上の管を保ちます。<ol>
<li>すぐに新鮮な生検の処理を開始します。</li>
	</ol>
</li>
	<li>凍結生検、フラッシュ チューブを含む生検を凍結し、使用するまで液体窒素で格納します。 注: 1 冷たい補われたバッファーが追加されるまで凍結サンプルを保つために重要です。</li>
</ol>2. バッファー準備
注: 始める前に、バッファーを次のとおり補足します。
<ol><li>1 mM DTT (最終濃度) を追加し、プロテアーゼとホスファターゼ阻害剤カクテル (最終濃度 x 1) 低張バッファー 1 および高張にバッファー 2 (表 1)。<ol>
<li>1 サンプルあたり 2 バッファーのバッファー 1、150 μ L の 1.5 mL を準備します。DTT は、サンプルの酸化を防ぐ。</li>
	</ol>
</li>
	<li>既に補われたバッファー 1 0.1% の最終濃度 1% 洗剤 (ノニル phenoxypolyethoxylethanol) を追加することによって核洗浄バッファーを準備します。1 mL のサンプルあたり核洗浄バッファーを準備します。</li>
</ol>3. 生検均質化
<ol><li>
新鮮な生検
	<ol>
<li>電動ミキサーで使い捨ての杵を配置します。杵が管の底に達することを確認します。</li>
		<li>Cryotube から、生検を取り出し、ピペット チップを 1.5 mL チューブに配置。</li>
		<li>1.5 mL チューブに補われたバッファー 1 の 200 μ L を追加します。</li>
		<li>杵で生検を均質化組織は完全に崩壊するまで氷の上保管してください。</li>
		<li>すべてのバイオプシーが均質化した後は、手順 4 に進みます。 注: 使い捨てすりこぎは、バイオプシーの間変更する必要があります。</li>
	</ol>
</li>
	<li>
冷凍生検 注意: 慎重に液体窒素を取り扱い皮膚や目に液体窒素との接触は、凍結重傷を引き起こす可能性があります。液体窒素を使用するときに常に手や目を保護します。<ol>
<li>いくつかの液体窒素を窒素タンクから取得した cryotubes を保存するボックスで取る。</li>
		<li>生検は、窒素タンクと液体窒素でボックスに、cryotube を配置します。すべての生検のためには、この手順を繰り返します。</li>
		<li>1.5 mL チューブに、cryotube から生検を転送します。 注: 生検は凍結チューブに添付されます。それは管から接続解除されますので滅菌ピペット チップで生検をプッシュします。</li>
		<li>補われたバッファー 1 の 200 μ L を追加します。</li>
		<li>乳棒と乳鉢を使用して、組織は完全に崩壊するまで、すべてのプロシージャ全体で 4 ° C の温度を維持する組織をホモジナイズしてください。 注: バッファーを追加する前に解凍生検をてはいけないです。</li>
	</ol>
</li>
</ol>4. 細胞質の分離
<ol><li>10 分間氷の上の均質化の生検を孵化させなさい。</li>
	<li>各サンプルを最終濃度 0.05% に 1% 洗剤 (ノニル phenoxypolyethoxylethanol) の 10 μ L を追加します。 注: セルにうねり、浸透圧換散によってバースト中性洗剤は細胞膜を破壊します。</li>
	<li>氷で 5 分間インキュベートします。</li>
	<li>簡単に渦と 4 ° C で 400 x g で 2 分間遠心します。</li>
	<li>ボルテックス後、上澄みを除去し、新しいきれいな 1.5 mL チューブ; に転送これは細胞質画分になります。ペレットは捨てないでください。</li>
</ol>5. 核隔離
<ol><li>核洗浄バッファーの 200 μ L で前の手順からペレットを再懸濁します。</li>
	<li>400 x g と 4 ° C で 2 分間チューブを遠心分離します。</li>
	<li>上澄みを廃棄し、さらに 2 回洗濯の手順を繰り返します。 注: 核の細胞質の汚染は、洗浄が削除されます。</li>
	<li>第 3 洗浄後バッファー 2 の 100 μ L にペレットを再懸濁します。</li>
	<li>また 30 分の 4 ° C でサンプルを積極的に振る、氷と渦 5 分ごとにサンプルをインキュベートします。</li>
	<li>後揺れ、トップ スピードで 10 分間のサンプルを遠心分離機 (> 12,000 g x) および 4 ° C</li>
	<li>ボルテックス後、新しいきれいな 1.5 に上清を移します。mL のチューブ。これは、核分画になります。</li>
</ol>6. プロテインの定量
注: 定量化ビシンコニン酸 (BCA) タンパク質アッセイ キットとタンパク質です。分数の蛋白質の濃度が範囲 0.5 ~ 5 μ g/μ L、核の一部分に低く (2 mg 生検から得られた蛋白質濃度の表 2を参照)。
<ol><li>次のウシ血清アルブミン (BSA) 量 (μ g)12の標準曲線の準備: 0-0.5-1-2-3-4-5-6。</li>
	<li>サンプルあたり BCA ファイナルミックスの 100 μ L を使用するために必要な試薬を準備します。</li>
	<li>96 ウェル プレートに各蛋白分画の 2 μ L をピペット BCA ミックスを追加して、読書のための製造元の指示に従ってください。</li>
</ol>

<ol>
	<li>Cox, B., Emili, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+subcellular+fractionation+and+protein+extraction+for+use+in+mass-spectrometry-based+proteomics.">Tissue subcellular fractionation and protein extraction for use in mass-spectrometry-based proteomics.</a> Nature Protocols. 1 (4), 1872-1878 (2006).</li><li>Itzhak, D. N., Tyanova, S., Cox, J., Borner, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global,+quantitative+and+dynamic+mapping+of+protein+subcellular+localization.">Global, quantitative and dynamic mapping of protein subcellular localization.</a> eLife. 5, (2016).</li><li>Preedy, V. R., Watson, R. R., Ronald, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in disease investigating the gastrointestinal tract. , (1998).</li><li>Alex, P., Gucek, M., Li, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+proteomics+in+the+study+of+inflammatory+bowel+diseases:+Current+status+and+future+directions+with+available+technologies.">Applications of proteomics in the study of inflammatory bowel diseases: Current status and future directions with available technologies.</a> Inflammatory bowel diseases. 15 (4), 616-629 (2009).</li><li>Ericsson, C., Franz&#233;n, B., Nist&#233;r, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frozen+tissue+biobanks.+Tissue+handling,+cryopreservation,+extraction,+and+use+for+proteomic+analysis.">Frozen tissue biobanks. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Mammalian+Organelle+Map+by+Protein+Correlation+Profiling.">A Mammalian Organelle Map by Protein Correlation Profiling.</a> Cell. 125 (1), 187-199 (2006).</li><li>Baghirova, S., Hughes, B. G., Hendzel, M. J., Schulz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+fractionation+and+isolation+of+subcellular+proteins+from+tissue+or+cultured+cells.">Sequential fractionation and isolation of subcellular proteins from tissue or cultured cells.</a> MethodsX. 2, e440-e445 (2015).</li><li>B&#246;rner, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+protein+extraction+from+human+pancreatic+cancer+tissues.">Subcellular protein extraction from human pancreatic cancer tissues.</a> BioTechniques. 46 (4), 297-304 (2009).</li><li>Antalis, T. M., Shea-Donohue, T., Vogel, S. 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著者が明らかに何もありません。

ここで説明したプロトコルは腸生検の核および細胞質分別のため使用されます。浄化された蛋白質が変性していないとだけでなく、免疫沈降、電気泳動の移動性シフト試金 (EMSA) などのネイティブ折り畳まれたタンパク質を必要とするアッセイにも図 2と3に示されて西部のしみの分析で使用できますかネイティブ ポリアクリルアミドゲル電気泳動 (ページ)?...

蛋白質はセル内のほぼすべての生物学的機能に参加し、構成、量や場所の変化は、病原性のシナリオにつながることができます。組織サンプルの分別方法は病気の複雑さを軽減する有用なアプローチ関連タンパク質解析。いくつかの研究蛋白質ローカライズ情報を使用または特定の細胞内コンパートメントからタンパク質を豊かにそのままな蛋白質の再現可能な分別のためのプロトコルは特定の生物的質問に答えるために役立ちます。タンパク質の細胞内局在を決定し、彼らのコンパートメントの再配布または基底での相互作用を監視病態の疾患の識別に役立つ、関連する機能の違い1,2。したがって、このメソッドは、再現性をもって分別腸組織生検、新鮮、冷凍、細胞質と核の細胞内コンパートメントを目指しています。
腸生検内視鏡手順3 (図 1) 日常的に得られる、タンパク質定量や免疫沈降法研究の効果的に使用できます。疾患病態4に直接かかわることができる蛋白質、腸上皮組織試料に含まれるので腸生検サンプル、腸疾患固有の成功の識別のための非常に貴重な情報源蛋白質。タンパク質解析のための有用な資源であり、シンプルで再現性のある試料は制限量を使用して細胞コンパートメント情報を提供するためにキー問題臨床情報と共にストアド冷凍、患者組織サンプルを凍結組織5。
細胞分画の分離のためのいくつかの市販キットがありますが、これらはより高価で、一般的に時間がかかるここで提示されたプロトコルよりも。多段勾配遠心法に基づいてプロトコルおよび連続的な勾配はまた以前使用された異なったティッシュ6、7の多様な細胞コンパートメントの分別のため。連続的な勾配の一貫性を維持することは、しばしば困難な作業です。膜の連続溶解に基づく類似したプロトコルが以前に記載されているが、彼らは一般的に 3 つ以上のバッファーを必要し、時間に手が長く8 。
組織サンプルを分留するとき覚えておいてその組織培養細胞の存在も重要な蛋白質の抽出に進む、サンプル プレゼント細胞間や細胞-マトリックスの対話。細胞と細胞外基質タンパク質の質に影響を与えることがなく適切な内訳を腸組織タンパク質分離9で重要な要因となります。このプロトコルでセルとセル行列連絡先最初壊れている、セルを解放し、個々 のセルに到達するバッファーを許可します。タンパク質コンパートメント分別に混合を避けるためには、細胞や原子膜の完全性を変えることがなく連絡先の内訳があります。
による小腸粘膜10様々 なプロテアーゼの濃縮、抽出中にタンパク質の分解を制御することが重要です。潜在的なタンパク質の分解を抑えるタンパク質抽出バッファーに複数のプロテアーゼ阻害剤を含める必要があります。さらに、抽出機能アッセイに使用する場合は、このタンパク質活動11の損失が発生、タンパク質やタンパク質の変性を避けるために不可欠です。このため、プロトコルは 4 ° C で行われた、新鮮な特異フッ化物 (PMSF) が 0.1 mM を使って、さらにタンパク質分解を阻害する直前の最終濃度でバッファーに追加されます。
セル分別の最初のステップは、組織の混乱とセル換散です。前述のように、目的は細胞とそれらを最小限のダメージでこじ開けるを細かく分けたです。腸の組織サンプルを均質化する必要がありますと、細胞が細胞膜の最大破壊を達成するために分離します。このプロトコルでは数秒以内に高速ボルテックス アクションによる均質化は腸の組織を分割するのにモーターを備えられた杵ミキサーを使用します。均質化後、細胞に細胞膜を破裂が、核をそのまま維持、非変化洗剤 (ノニル phenoxypolyethoxylethanol) と核を分離する短い渦の加え、続いて低張バッファーで孵化します。細胞質分画。細胞核は細胞質を除去した後揺れで高張バッファーどっと。
ここで紹介した方法は、内視鏡手術と重 2 と 10 mg の間の範囲の間に得られた生鮮・冷凍の腸生検の細胞レベル下の分別のための適切なメソッドです。プロトコルは簡単で再現性のある、基本的な実験装置および時間の下で試薬を使用して実行可能性があります。

<table border="0" cellpadding="0" cellspacing="0" style="width:657px;" width="658">
	<tbody>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">HEPES</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:119px;">H4034-1kg</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">KCl</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:119px;">P9333-1kg</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">EDTA</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:119px;">E9884-100G</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">NaCl</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:119px;">5588886-1kg</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">DTT</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:119px;">10197777001</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">PMSF</td>
			<td style="width:128px;">Thermo Scientific</td>
			<td style="width:119px;">36978</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">Proteinase and phosphatase inhibitors</td>
			<td style="width:128px;">Thermo Scientific</td>
			<td style="width:119px;">A32959</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">NP-40&#160;</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:119px;">CA-630</td>
			<td>Detergent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">BCA assay</td>
			<td style="width:128px;">Thermo Scientific</td>
			<td style="width:119px;">23227</td>
			<td>Protein quantification kit</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">Disposable plastic pestles</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:119px;">Z359947-100EA</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">Dounce homogeneizer</td>
			<td style="width:128px;">VWR</td>
			<td style="width:119px;">431-0100</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">Microcentrifuge</td>
			<td style="width:128px;">Eppendorf</td>
			<td style="width:119px;">5415-R</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:244px;">Shacker</td>
			<td style="width:128px;">IKA</td>
			<td style="width:119px;">MS3 basic</td>
			<td></td>
		</tr>
	</tbody>
</table>

subcellular fractionation from fresh and frozen gastrointestinal specimens

このプロトコルの目的は特定の蛋白質または異なる組織状態 (すなわち、健全な蛋白質の複合体の局在解析用核と細胞質コンパートメントに内視鏡検査によって得られる人間の腸組織を分別するには対病)。このメソッドは両方生鮮・冷凍の腸組織サンプルの分別のため便利簡単にアクセスできるすべての研究所、いない時間がかかるです。

図 1は、ヘマトキシリン エオシン染色腸生検部そのまま絨毛と地下構造を示しています。
このプロトコルを使用して核および細胞質分別の代表の結果は、図 2および3に示します。冷凍 (Fz) フレッシュ (Fh)、図 2に示すように実験で生検はここで提示され...

Watch this Scientific Journal Video about Subcellular Fractionation from Fresh and Frozen Gastrointestinal Specimens at JoVE.com

Subcellular Fractionation from Fresh and Frozen Gastrointestinal Specimens

ここでは、人間および冷凍腸生検で細胞質および核蛋白質の細胞レベル下の分離の簡単な細胞分画を実行するプロトコルを提案する.

生鮮・冷凍の消化試験片から細胞レベル下の分別

The purpose of this protocol is to fractionate human intestinal tissue obtained by endoscopy into nuclear and cytoplasmic compartments for the ...

subcellular-fractionation-from-fresh-frozen-gastrointestinal

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Medical-Surgical Nursing

Immunology and Infection

Unit 1

An Overview of the Gastrointestinal System: Anatomy, Physiology, and Assessment

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9.7K ビュー.  University of the Basque Country (UPV-EHU), Biocruces Health Research Institute. ここでは、人間および冷凍腸生検で細胞質および核蛋白質の細胞レベル下の分離の簡単な細胞分画を実行するプロトコルを提案する.

ビデオ: Subcellular Fractionation from Fresh and Frozen Gastrointestinal Specimens

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

粘着テープベースのサンプリングと蛍光の組み合わせその場で</emの迅速検出のための>ハイブリダイゼーションサルモネラ生鮮食品について

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

免疫・感染学

Competitive Homing Assays to Study Gut-tropic T Cell Migration

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial cells and tissue extravasation is a multistep process controlled by different adhesion molecules (homing receptors) expressed on lymphocytes and their respective ligands (addressins) displayed on endothelial cells 1 2. Even though the function of these adhesion receptors can be partially studied ex vivo, the ultimate test for their physiological relevance is to assess their role during in vivo lymphocyte adhesion and migration. Two complementary strategies have been used for this purpose: intravital microscopy (IVM) and homing experiments. Although IVM has been essential to define the precise contribution of specific adhesion receptors during the adhesion cascade in real time and in different tissues, IVM is time consuming and labor intensive, it often requires the development of sophisticated surgical techniques, it needs prior isolation of homogeneous cell populations and it permits the analysis of only one tissue/organ at any given time. By contrast, competitive homing experiments allow the direct and simultaneous comparison in the migration of two (or even more) cell subsets in the same mouse and they also permit the analysis of many tissues and of a high number of cells in the same experiment.
Here we describe the classical competitive homing protocol used to determine the advantage/disadvantage of a given cell type to home to specific tissues as compared to a control cell population. We chose to illustrate the migratory properties of gut-tropic versus non gut-tropic T cells, because the intestinal mucosa is the largest body surface in contact with the external environment and it is also the extra-lymphoid tissue with the best-defined migratory requirements. Moreover, recent work has determined that the vitamin A metabolite all-trans retinoic acid (RA) is the main molecular mechanism responsible for inducing gut-specific adhesion receptors (integrin a4b7and chemokine receptor CCR9) on lymphocytes. Thus, we can readily generate large numbers of gut-tropic and non gut-tropic lymphocytes ex vivoby activating T cells in the presence or absence of RA, respectively, which can be finally used in the competitive homing experiments described here.

Competitive homing experiments allow to directly assessing the migratory properties of two different cell populations in a single mouse. Here we illustrate this procedure by comparing the migration of ex vivo-generated gut-tropic versus non-gut tropic T cells.

腸管指向性T細胞の移動を研究するために競争力のあるホーミングアッセイ

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial ...

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

のRNAの単離緑膿菌マウス消化管に定着

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

Optimized Protocols for Mycobacterium leprae Strain Management: Frozen Stock Preservation and Maintenance in Athymic Nude Mice

Leprosy, caused by Mycobacterium leprae, is an important infectious disease that is still endemic in many countries around the world, including Brazil. There are currently no known methods for growing M. leprae in vitro, presenting a major obstacle in the study of this pathogen in the laboratory. Therefore, the maintenance and growth of M. leprae strains are preferably performed in athymic nude mice (NU-Foxn1nu). The laboratory conditions for using mice are readily available, easy to perform, and allow standardization and development of protocols for achieving reproducible results. In the present report, we describe a simple protocol for purification of bacilli from nude mouse footpads using trypsin, which yields a suspension with minimum cell debris and with high bacterial viability index, as determined by fluorescent microscopy. A modification to the standard method for bacillary counting by Ziehl-Neelsen staining and light microscopy is also demonstrated. Additionally, we describe a protocol for freezing and thawing bacillary stocks as an alternative protocol for maintenance and storage of M. leprae strains.

Optimized Protocols for Mycobacterium leprae Strain Management: Frozen Stock Preservation and Maintenance in Athymic Nude Mice

Mycobacterium leprae, the causative agent of leprosy, does not grow in vitro. We describe an easy to follow protocol to prepare a bacillary suspension to ensure the maintenance of large quantities of M. leprae for a variety of applications. Protocols for propagation by mouse footpad inoculation, evaluation of viability, freezing and thawing bacillary stock are described in detail.

のための最適化されたプロトコル<em&gt;らい菌</em&gt;ひずみ管理：無胸腺ヌードマウスにおける凍結ストック保全とメンテナンス

Leprosy, caused by Mycobacterium leprae, is an important infectious disease that is still endemic in many countries around the world, including Brazil. ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

簡単かつ迅速なプロトコル非酵素的に浸潤リンパ球の分析のための新鮮なヒト組織を解離

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

エフェクターCD4の養子移入によるマウス腸の炎症の誘導<sup&gt; +</sup&gt; CD45RB<sup&gt;高い</sup&gt;免疫不全マウスへのT細胞

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat gastrointestinal disorders. Adapting probiotic yeast in particular for this approach carries several advantages. These strains have not only the potential to synthesize a wide variety of complex heterologous proteins but are also capable of surviving and protecting those proteins during transit through the intestine. Critically, however, this approach requires expertise in many diverse laboratory techniques not typically used in tandem. Furthermore, although individual protocols for yeast transformation are well characterized for commonly used laboratory strains, emphasis is placed here on alternative approaches and the importance of optimizing transformation for less well characterized probiotic strains. Detailing these methods will help facilitate discussion as to the best approaches for testing probiotic yeast as oral drug delivery vehicles and indeed serve to advance the development of this novel strategy for gastrointestinal therapy.

Presented here is a unified description of techniques that can be used to develop, transform, administer, and test heterologous protein expression of the probiotic yeast Saccharomyces boulardii.

マウスにおける強制経口投与に続いてプロバイオティクス酵母の形質転換および消化管免疫組織から彼らの回復

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

ナノスケールPCRによる動物標本における呼吸器病原体のハイスループット検出

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence microscopy as observations made at a population level may not truly reflect what occurs at a single cell level. Live-cell timelapse microscopy allows investigators to monitor the changes in cell division or cell morphology which provide valuable insights regarding subcellular localization of proteins and timing of gene expression, as it happens, to potentially aid in answering important biological questions. Here, we describe our protocol to monitor phenotypic changes in Bacillus subtilis and Staphylococcus aureus using a high-resolution deconvolution microscope. The objective of this report is to provide a simple and clear protocol that can be adopted by other investigators interested in conducting fluorescence microscopy experiments to study different biological processes in bacteria as well as other organisms.

This article provides a step-by-step guide to investigate protein subcellular localization dynamics and to monitor morphological changes using high-resolution fluorescence microscopy in Bacillus subtilis and Staphylococcus aureus.

細菌における細胞内タンパク質の局在化と細胞形態の変化を調べる生細胞蛍光顕微鏡

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence ...

Isolation of Macrophage Subsets and Stromal Cells from Human and Mouse Myocardial Specimens

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative responses after cardiac injury. How various subsets of macrophages orchestrate the immune responses after cardiac injury is an active area of research. Presented here is a simple protocol that our lab performs routinely, for the extraction of macrophages from mouse and human myocardium specimens obtained from healthy and diseased individuals. Briefly, this protocol involves enzymatic digestion of cardiac tissue to generate a single cell suspension, followed by antibody staining, and flow cytometry. This technique is suitable for functional assays performed on sorted cells as well as bulk and single cell RNA sequencing. A major advantage of this protocol is its simplicity, minimal day to day variation and wide applicability allowing investigation of macrophage heterogeneity across various mouse models and human disease entities.

Presented here is a protocol to isolate various subsets of macrophages and other non-immune cells from human and mouse myocardium by preparing a single cell suspension through enzymatic digestion. Gating schemes for flow cytometry based identification and characterization of isolated macrophages are also presented.

ヒトおよびマウスの心筋標本からのマクロファージサブセットおよび間質細胞の分離

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative ...