NIH（1 K08 DK080221 - 01）とヴァンダービルト制度開発基金から提供された資金。

パート1：傷害 - 修復大腸炎モデル<ol><li>ベースラインの重量は、DSSの治療を開始する前に、各マウスのために取得する必要があります。 </li><li> 3％（w / v）のデキストラン硫酸ナトリウム塩の水溶液と0.45μmのセルロースアセテートフィルターでフィルターを作成します。 </li><li>四日間のための3％DSS溶液を用いてマウスのケージに飲料水を交換してください。マウスは、水の他のソース（すなわち、排除のヒントは、自動給水システム上に配置）へのアクセスを持つべきではありません。 </li><li> 4日目に、いくつかの結腸上皮の回復を可能に、さらに3日間、水とDSSソリューションを交換してください。マウスは大腸炎の全身的影響を定量化するために4日目に体重を測定する必要があります。体重減少は深刻な損傷と共通です。 </li><li> 7日目に、マウスの重量を測定し、生け贄に捧げる。マウスは、イソフルラン、または他の制度の吸入過剰投与により安楽死することができる、IACUCは方法を承認した。 </li></ol>パート2：コロンの剖検と収穫<ol><li>腹部への遮るもののないアクセスを確保するために、マウスで安全な脚の腹側を公開します。 70％エタノールで完全に腹部を濡らす。 </li><li>組織鉗子による腹部の正中線をつかみ、テン肌をこのように上向きに延長する。腹部の内容を公開するため、腹部を切開するために先の細いはさみを使用してください。正中線でxyphoidプロセスの先端に切開を延長し、二国間沿岸マージンの下面に沿って延びている。 </li><li>小腸、盲腸、結腸を識別します。周囲の腸間膜からコロンを慎重に解剖/いじめる。遠位結腸と直腸を解放するために骨盤内の深いコロンを横に切断する。近位結腸を解放するcolonocecalマージンでコロンを横に切断する。ケアは、穿孔が発生した場合にコロンが問題となる洗浄としてコロンを傷つけないように、このプロセス中に注意する必要があります。 </li><li> 20G送り針と10mlのシリンジを使用して、挿管と溶出液が糞便の完全に透明になるまで氷冷したPBSでコロンをフラッシュする。 </li><li>解剖顕微鏡で肉眼的分析が必要な場合は、この時点で、、コロンは&quot;ソーセージ&quot;として固定することができます。もし組織学的分析は、&quot;急性大腸炎の組織学的分析のためにスイスロールを作る&quot;第4部に進むよりも望まれている。 </li></ol>パート3：結腸全体の巨視的分析のための&quot;ソーセージ&quot;として処理<ol><li>コロンの正しい向きを維持することが重要である、従って、オペレータに最も近い大腸の遠位端を保持。非吸収性縫合糸の2つの部分（SP116 1.5メトリック編みシルク）、長さは1約1インチ、他の2インチカット。 </li><li>それでも良好なシール性を維持しながら可能な限り余裕の近くに遠位端をオフに結びつけるため縫合の2インチの部分を使用してください。 </li><li> 10％の5mlを含む20G送り針が大腸の近位端にリン酸ホルマリンをバッファに置きます。疎供給針の球のすぐ近位縫合糸の残りの部分を結ぶ。供給針の電球でしっかりとコロンをつかんで、コロンが展開されるまで、ホルマリン注入する。あなたがこのように、&quot;ソーセージ&quot;として注入された、拡張されたコロンを残して、摂食針を撤回として縫合糸の結び目を締めます。 </li><li> 10％がリン酸ホルマリンをバッファリングし、チューブにコロンを置くと15 mlコニカルチューブを埋める。 24時間を修正しました。 </li><li>ホルマリンを捨て、追加の24時間、70％EtOHで交換してください。 （コロンは、室温で無期限に70％エタノールで保存することができます。） </li><li>コニカルチューブからコロンを削除し、コロンを傷つけないように注意しながら、メスで両端にある文字列をカット。遠位端は、文字列の最長の部分を持っていることに注意してください。それは長いシートを形成するように遠位から結腸の近位端に縦にカット。この時点で、コロンは、解剖顕微鏡下で見ることができます。 </li></ol>パート4：急性大腸炎の組織学的分析のためのスイスロールの作成<ol><li>コロンの正しい向きを維持することが重要である、従って、オペレータに最も近い大腸の遠位端を保持。 </li><li>大腸の長さを測定します。このメトリックは、傷害の重症度の別の指標です。大腸炎は、浮腫が増加し、全体的な大腸の長さが短くなります。 </li><li>先の細いはさみを使用して、遠位から結腸の近位端に縦に切開する。このように平らなシートとしてコロンを表示する、切開のいずれかの端を持ち、遠位 - &gt;近位に、あなたのように働く横方向に開くように微細な先端鉗子を使用してください。 </li><li>コロンローリングピンセットと両手のテクニックが必要です。鉗子と遠位マージンのいずれかの端をつかみます。順次近位縁に進む鉗子を回転させ、解放することによって大腸をロールに進んでください。従って、三次元または&quot;スイスロール&quot;で螺旋を生成する。ロール状を維持するために、鉗子でしっかりとロールを把握し、27G1 / 2針でそれを横に切断する。それが終了するロールを次のように針を曲げて針を固定します。 </li><li> 10％の瓶で適切に標識された組織のカセットと場所でロールを配置するには、組織の固定を確実にするために24時間室温でリン酸ホルマリン緩衝化。 </li><li>ホルマリンを捨て、追加の24時間、70％EtOHで交換してください。 （コロンは、室温で無期限に70％エタノールで保存することができます。） </li><li>サンプルを修正したら、それはパラフィン包埋、切片と組織学的分析のためのスライド上にマウントすることができます。 </li></ol>パート5：代表的な結果急性大腸炎のためのDSSモデルは、研究者は、取得する修正、およびそのモデル急性大腸炎大腸を分析することができます。 （図1）適切にロールバックならスイスロールをカットしてマウントされると、それは全体のコロンの代表的なスライスを形成すべきである。マウントされているロールは、遠位（内側）の端から近位（外側）の端（図2）に、コロンに損傷の程度を決定するために、H＆Eで染色することができます。免疫組織化学はまた、炎症性細胞浸潤を同定および定量するためにスイスロールで実行することができます。ソーセージのメソッドが正しく実行されている場合は、固定されたコロンは瞳孔が開いされ、全体の粘膜表面は、容易に改ざんし、解剖顕微鏡（図3）の下に表示することができます。 <a href="https://www.jove.com/files/ftp_upload/1652/1652fig1.jpg"><img src="/files/ftp_upload/1652/1652fig1tmb.jpg" alt="図1" border="0" /></a> 図1適切に実行される&quot;スイスロール&quot;。 （A）コロンは、近位端に遠位からロール針でtransectedと針の端を折り曲げて固定されています。その後、固定のために組織カセットに配置されます。 （B）H＆Eは、DSS（D =遠位結腸P =近位結腸）で治療したマウスの結腸で作られたスイスロールの5μmのセクションを染色した。 <a href="https://www.jove.com/files/ftp_upload/1652/1652fig2.jpg"><img src="/files/ftp_upload/1652/1652fig2tmb.jpg" alt="図2" border="0" /></a> 図2：DSS扱わコロンは急性大腸炎の徴候を示す。炎症と陰窩の損傷は、水処理対照と比較してDSS処理コロンで明白である。 <a href="https://www.jove.com/files/ftp_upload/1652/1652fig3.jpg"><img src="/files/ftp_upload/1652/1652fig3tmb.jpg" alt="図3" border="0" /></a> 図3。&quot;ソーセージ&quot;の例。ソーセージは、ホルマリンを注入し、完全に拡大した。わずかな角度は、コロンの自然な湾曲に存在し、セカンダリになります。開かれたソーセージは、平らになるように置いてください。

<ol>
	<li>Okayasu, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+in+the+induction+of+reliable+experimental+acute+and+chronic+ulcerative+colitis+in+mice.">A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice.</a> Gastroenterology. 98 (3), 694-702 (1990).</li><li>Martinez, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+Mtgr1+sensitizes+the+colonic+epithelium+to+dextran+sodium+sulfate-induced+colitis.">Deletion of Mtgr1 sensitizes the colonic epithelium to dextran sodium sulfate-induced colitis.</a> Gastroenterology. 131 (2), 579-588 (2006).</li><li>Moolenbeek, C., Ruitenberg, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+"Swiss+roll":+a+simple+technique+for+histological+studies+of+the+rodent+intestine.">The "Swiss roll": a simple technique for histological studies of the rodent intestine.</a> Lab Anim. 15 (1), 57-59 (1981).</li><li>Mahler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+susceptibility+of+inbred+mouse+strains+to+dextran+sulfate+sodium-induced+colitis.">Differential susceptibility of inbred mouse strains to dextran sulfate sodium-induced colitis.</a> Am J Physiol. 274, G544-551 (1998).</li><li>Aharoni, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunomodulatory+therapeutic+effect+of+glatiramer+acetate+on+several+murine+models+of+inflammatory+bowel+disease.">Immunomodulatory therapeutic effect of glatiramer acetate on several murine models of inflammatory bowel disease.</a> J Pharmacol Exp Ther. 318 (1), 68-78 (2006).</li><li>Hahm, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+transforming+growth+factor+beta+signalling+in+the+intestine+contributes+to+tissue+injury+in+inflammatory+bowel+disease.">Loss of transforming growth factor beta signalling in the intestine contributes to tissue injury in inflammatory bowel disease.</a> Gut. 49 (2), 190-198 (2001).</li><li>Green, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adrenergic+modulation+of+Escherichia+coli+O157:H7+adherence+to+the+colonic+mucosa.">Adrenergic modulation of Escherichia coli O157:H7 adherence to the colonic mucosa.</a> Am J Physiol Gastrointest Liver Physiol. 287 (6), G1238-1246 (2004).</li><li>Dieleman, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+experimental+colitis+induced+by+dextran+sulphate+sodium+(DSS)+is+characterized+by+Th1+and+Th2+cytokines.">Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines.</a> Clin Exp Immunol. 114 (3), 385-391 (1998).</li></ol>

動物実験は、ヴァンダービルトIACUCによって定められたガイドラインおよび規制に準拠して行った。

このプロトコルは、急性損傷、傷害、修復、または慢性の結腸傷害のプロセスをモデル化するように変更することができます。 急性傷害の変更： 即時の犠牲に続いて4-6日のためのDSSの治療のアドリブ傷害修復の変更： 3-4日と犠牲の回復期間（上記のプロトコルで説明されている）に続いてDSS治療の4-6日で負傷?...

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		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>Dextran Sulfate Sodium Salt</td>
<td>&nbsp;</td>
<td>USB Corp</td>
<td>9011-18-1</td>
<td>mol wt 40,000-50,000</td>
</tr>
<tr>
<td>10% Buffered Formalin Phosphate</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>SF100-4</td>
<td>&nbsp;</td>
<tr>
<td>Isoflurane, USP</td>
<td>&nbsp;</td>
<td>Phoenix Pharmaceuticals</td>
<td>NDC 57319-474-06</td>
<td>&nbsp;</td>
<tr>
<td>TISSUE PATH Macrosette Cassettes</td>
<td>&nbsp;</td>
<td>Fisher Healthcare</td>
<td>15182706</td>
<td>&nbsp;</td>
<tr>
<td>BD 10 ml Syringe</td>
<td>&nbsp;</td>
<td>B-D</td>
<td>309604</td>
<td>&nbsp;</td>
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<td>Ethyl Alcohol</td>
<td>&nbsp;</td>
<td>Pharmco-Aaper</td>
<td>E190</td>
<td>Dilute to 70% with distilled water</td>
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<td>20G Straight Feeding Needle</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>20068-612</td>
<td>&nbsp;</td>
<tr>
<td>27G1/2 PrecisionGlide Needle</td>
<td>&nbsp;</td>
<td>B-D</td>
<td>305109</td>
<td>&nbsp;</td>
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<td>Dissecting Scissors Sharp/Blunt Tip</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>82027-588</td>
<td>&nbsp;</td>
<tr>
<td>Waugh Forceps</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>82027-428</td>
<td>&nbsp;</td>
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<td>Non-absorbable Suture </td>
<td>&nbsp;</td>
<td>LOOK Surgical Specialties</td>
<td>SP116</td>
<td>&nbsp;</td>
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<td>Whatman Blotting Paper</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>28298-020</td>
<td>Cut as needed</td>
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<td>Nalgene Surfactant-Free Cellulose Acetate (SFCA) Filter</td>
<td>&nbsp;</td>
<td>Cole-Parmer</td>
<td>EW-06731-2</td>
<td>&nbsp;</td>
<tr>
<td>Carbon fiber composites digital caliper</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>15-007-958</td>
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murine colitis modeling using dextran sulfate sodium (dss)

- クローン病大腸炎特に潰瘍性大腸炎およびクローン病の大腸亜種、大腸炎は、ウイルスや細菌感染症、虚血性傷害、または自己免疫疾患から発生する可能性があります。急性大腸炎は、腹痛や膨満、吸収不良、下痢、血便、便中の粘液を呈することがある。我々は環境、遺伝学、および大腸の炎症性腸疾患と動物モデルにおける上皮のバリア機能不全この疾患の我々の理解を進める上で不可欠であったとの間の複雑な相互作用を理解し始めている。一つの人気モデルは数日間続く上皮損傷と大腸で堅牢な炎症反応の結果、低分子量デキストラン硫酸ナトリウム（DSS）でマウスの飲料水を補完する関係<sup&gt; 1</supこのアプローチの&gt;。バリエーションは、急性損傷、修復が続く急性傷害、および回復のモデリングの慢性炎症性疾患を織り交ぜDSSの反復サイクルをモデル化するために使用することができます。<sup&gt; 2</sup&gt;。飲料水に3％DSSの1台の4日間の治療後、マウスは、体重減少、血便、下痢などの急性大腸炎の徴候を示す。マウスは、治療コースの終了時に安楽死され、剖検解剖コロンで処理され、&quot;スイスが&quot;ロールバックすることができます<sup&gt; 3</sup&gt;結腸全体の顕微鏡分析を許可または巨視的な分析を可能にするために&quot;ソーセージ&quot;としてホルマリンを注入する。組織は、その後パラフィン、切片、および組織学的な見直しのためのステンドグラスに組み込まれています。

Watch this Scientific Journal Video about Murine Colitis Modeling using Dextran Sulfate Sodium DSS at JoVE.com

Murine Colitis Modeling using Dextran Sulfate Sodium DSS

飲料水で投与デキストラン硫酸ナトリウム（DSS）は、急性大腸炎の確立されたマウス炎症性損傷モデルです。このプロトコルは、DSSの治療や組織の準備のための方法を概説します。

デキストラン硫酸ナトリウム（DSS）を用いたマウス大腸炎モデル

Colitis can occur from viral or bacterial infections, ischemic insult, or autoimmune disorders; most notably Ulcerative Colitis and the colonic variant of ...

murine-colitis-modeling-using-dextran-sulfate-sodium-dss

Research

JoVE Journal

Biology

Murine Colitis Modeling using Dextran Sulfate Sodium (DSS)

49.7K ビュー.  Vanderbilt University. 飲料水で投与デキストラン硫酸ナトリウム（DSS）は、急性大腸炎の確立されたマウス炎症性損傷モデルです。このプロトコルは、DSSの治療や組織の準備のための方法を概説します。

ビデオ: Murine Colitis Modeling using Dextran Sulfate Sodium DSS

Predicting the Effectiveness of Population Replacement Strategy Using Mathematical Modeling

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy.  Insight is given into how computational models can provide information on the population dynamics of mosquitoes and the spread of transposable elements through A. gambiae subspecies.  The ethical considerations of releasing genetically modified mosquitoes into the wild are discussed.

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy. Insight is given into how computational models can provide information on the population dynamics of mosquitoes and the spread of transposable elements through A. gambiae subspecies. The ethical considerations of releasing genetically modified mosquitoes into the wild are discussed.

数理モデルを使用して人口置換戦略の有効性を予測

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy.  ...

生物学

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling1 and pressure injection of traceable enzymes2 to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time3-5. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish6-8. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularily attractive tools for lineage labeling and to observe the migration of cells in vivo7. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence9. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole10. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well11. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed 12,13. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequenctly, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.

レーザーUncagableフルオレセインデキストランでゼブラフィッシュ細胞の系統ラベリング

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

Investigating Intestinal Inflammation in DSS-induced Model of IBD

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease (CD). Both UC and CD, when present in the colon, generate a similar symptom profile which can include diarrhea, rectal bleeding, abdominal pain, and weight loss.1 Although the pathogenesis of IBD remains unknown, it is described as a multifactorial disease that involves both genetic and environmental components.2
There are numerous and variable animal models of colonic inflammation that resemble several features of IBD. Animal models of colitis range from those arising spontaneously in susceptible strains of certain species to those requiring administration of specific concentrations of colitis-inducing chemicals, such as dextran sulphate sodium (DSS). Chemical-induced models of gut inflammation are the most commonly used and best described models of IBD. Administration of DSS in drinking water produces acute or chronic colitis depending on the administration protocol.3 Animals given DSS exhibit weight loss and signs of loose stool or diarrhea, sometimes with evidence of rectal bleeding.4,5 Here, we describe the methods by which colitis development and the resulting inflammatory response can be characterized following administration of DSS. These methods include histological analysis of hematoxylin/eosin stained colon sections, measurement of pro-inflammatory cytokines, and determination of myeloperoxidase (MPO) activity, which can be used as a surrogate marker of inflammation.6
The extent of the inflammatory response in disease state can be assessed by the presence of clinical symptoms or by alteration in histology in mucosal tissue. Colonic histological damage is assessed by using a scoring system that considers loss of crypt architecture, inflammatory cell infiltration, muscle thickening, goblet cell depletion, and crypt abscess.7 Quantitatively, levels of pro-inflammatory cytokines with acute inflammatory properties, such as interleukin (IL)-1&beta;, IL-6 and tumour necrosis factor (TNF)-&alpha;,can be determined using conventional ELISA methods. In addition, MPO activity can be measured using a colorimetric assay and used as an index of inflammation.8
In experimental colitis, disease severity is often correlated with an increase in MPO activity and higher levels of pro-inflammatory cytokines. Colitis severity and inflammation-associated damage can be assessed by examining stool consistency and bleeding, in addition to assessing the histopathological state of the intestine using hematoxylin/eosin stained colonic tissue sections. Colonic tissue fragments can be used to determine MPO activity and cytokine production. Taken together, these measures can be used to evaluate the intestinal inflammatory response in animal models of experimental colitis.

Experimental models of inflammatory bowel disease have allowed us to examine the complex innate and adaptive immune responses associated with pathogenesis. Using histological scoring, quantification of pro-inflammatory cytokines and myeloperoxidase activity, one can begin to assess these responses seen in inflammatory bowel disease.

IBDのDSS誘発モデルにおける腸炎の調査

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

細胞トラッキング染料を使用して細胞分裂監視のための最適化された染色と増殖モデリング方法

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Assessing Murine Resistance Artery Function Using Pressure Myography

Pressure myograph systems are exquisitely useful in the functional assessment of small arteries, pressurized to a suitable transmural pressure. The near physiological condition achieved in pressure myography permits in-depth characterization of intrinsic responses to pharmacological and physiological stimuli, which can be extrapolated to the in vivo behavior of the vascular bed. Pressure myograph has several advantages over conventional wire myographs. For example, smaller resistance vessels can be studied at tightly controlled and physiologically relevant intraluminal pressures. Here, we study the ability of 3rd order mesenteric arteries (3-4 mm long), preconstricted with phenylephrine, to vaso-relax in response to acetylcholine. Mesenteric arteries are mounted on two cannulas connected to a pressurized and sealed system that is maintained at constant pressure of 60 mmHg. The lumen and outer diameter of the vessel are continuously recorded using a video camera, allowing real time quantification of the vasoconstriction and vasorelaxation in response to phenylephrine and acetylcholine, respectively.
	To demonstrate the applicability of pressure myography to study the etiology of cardiovascular disease, we assessed endothelium-dependent vascular function in a murine model of systemic hypertension. Mice deficient in the &alpha;1 subunit of soluble guanylate cyclase (sGC&alpha;1-/-) are hypertensive when on a 129S6 (S6) background (sGC&alpha;1-/-S6) but not when on a C57BL/6 (B6) background (sGC&alpha;1-/-B6). Using pressure myography, we demonstrate that sGC&alpha;1-deficiency results in impaired endothelium-dependent vasorelaxation. The vascular dysfunction is more pronounced in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice, likely contributing to the higher blood pressure in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice. 
	Pressure myography is a relatively simple, but sensitive and mechanistically useful technique that can be used to assess the effect of various stimuli on vascular contraction and relaxation, thereby augmenting our insight into the mechanisms underlying cardiovascular disease.

In pressure myography, an intact small segment of a vessel is mounted onto two small cannulas and pressurized to a suitable luminal pressure. Here, we describe the method to measure vasorelaxation response of the mouse 3rd order mesenteric arteries in c57 and sGC&alpha;1-/- mice using pressure myography.

圧力筋運動記録法を用いたマウス抵抗動脈機能を評価

Pressure myograph systems are  exquisitely useful in the functional assessment of small arteries, pressurized  to a suitable transmural pressure. The  ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

ゼブラフィッシュを用いた病的ヒトゲノムの in vivoモデルで

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated exocytosis is fundamental for intercellular communication and is a key mechanism for the secretion of neurotransmitters, hormones, inflammatory mediators, and other compounds, by a variety of cells. At least three distinct mechanisms are known for regulated exocytosis: full exocytosis, where a single SG fully fuses with the plasma membrane, kiss-and-run exocytosis, where a single SG transiently fuses with the plasma membrane, and compound exocytosis, where several SGs fuse with each other, prior to or after SG fusion with the plasma membrane. The type of regulated exocytosis undertaken by a cell is often dictated by the type of secretory trigger. However, in many cells, a single secretory trigger can activate multiple modes of regulated exocytosis simultaneously. Despite their abundance and importance across cell types and species, the mechanisms that determine the different modes of secretion are largely unresolved. One of the main challenges in investigating the different modes of regulated exocytosis, is the difficulty in distinguishing between them as well as exploring them separately. Here we describe the use of fluorescein isothiocyanate (FITC)-dextran as an exocytosis reporter, and live cell imaging, to differentiate between the different pathways of regulated exocytosis, focusing on compound exocytosis, based on the robustness and duration of the exocytic events.

Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically.

規制エキソサイトーシスの記者として FITC デキストランをイメージング

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated ...

In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy

Differentiation and maturation of megakaryocytes occur in close association with the cellular and extracellular components of the bone marrow. These processes are characterized by the gradual appearance of essential structures in the megakaryocyte cytoplasm such as a polyploid and polylobulated nucleus, an internal membrane network called demarcation membrane system (DMS) and the dense and alpha granules that will be found in circulating platelets. In this article, we describe a standardized protocol for the in situ ultrastructural study of murine megakaryocytes using transmission electron microscopy (TEM), allowing for the identification of key characteristics defining their maturation stage and cellular density in the bone marrow. Bone marrows are flushed, fixed, dehydrated in ethanol, embedded in plastic resin, and mounted for generating cross-sections. Semi-thin and thin sections are prepared for histological and TEM observations, respectively. This method can be used for any bone marrow cell, in any EM facility and has the advantage of using small sample sizes allowing for the combination of several imaging approaches on the same mouse.

In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy

Here, we present a protocol to analyze ultrastructure of the megakaryocytes in situ using transmission electron microscopy (TEM). Murine bone marrows are collected, fixed, embedded in epoxy resin and cut in ultrathin sections. After contrast staining, the bone marrow is observed under a TEM microscope at 120 kV.

イン・シトゥ 透過電子顕微鏡を用いたネズミ巨核生物の探索

Differentiation and maturation of megakaryocytes occur in close association with the cellular and extracellular components of the bone marrow. These ...

Assessment of Gut Barrier Integrity in Mice Using Fluorescein-Isothiocyanate-Labeled Dextran

Gut barrier integrity is a hallmark of intestinal health. While gut barrier integrity can be assessed using indirect markers such as the measurement of plasma inflammatory markers and bacterial translocation to the spleen and lymph nodes, the gold standard directly quantifies the ability of selected molecules to traverse the gut mucosal layer toward systemic circulation. This article uses a non-invasive, cost-effective, and low-burden technique to quantify and follow in real time the intestinal permeability in mice using fluorescein-isothiocyanate-labeled dextran (FITC-dextran). Prior to oral supplementation with FITC-dextran, the mice are fasted. They are then gavaged with FITC-dextran diluted in phosphate-buffered saline (PBS). One hour after the gavage, the mice are subjected to general anesthesia using isoflurane, and the in vivo fluorescence is visualized in an imaging chamber. This technique aims to assess residual fluorescence in the abdominal cavity and the hepatic uptake, which is suggestive of portal migration of the fluorescent probe. Blood and stool samples are collected 4 h after oral gavage, and the mice are sacrificed. Plasma and fecal samples diluted in PBS are then plated, and the fluorescence is recorded. The concentration of FITC-dextran is then calculated using a standard curve. In previous research, in vivo imaging has shown that fluorescence rapidly spreads to the liver in mice with a weaker gut barrier induced by a low-fiber diet, while in mice supplemented with fiber to strengthen the gut barrier, the fluorescent signal is retained mostly in the gastrointestinal tract. In addition, in this study, control mice had elevated plasma fluorescence and reduced fluorescence in the stool, while inversely, inulin-supplemented mice had higher levels of fluorescence signals in the gut and low levels in the plasma. In summary, this protocol provides qualitative and quantitative measurements of intestinal permeability as a marker for gut health.

In the present study, fluorescein-isothiocyanate-labeled (FITC) dextran is administered to mice via oral gavage to evaluate intestinal permeability both in vivo and in plasma and fecal samples. As gut barrier function is affected in many disease processes, this direct and quantitative assay can be used in diverse research areas.

フルオレセインイソチオシアネート標識デキストランを用いたマウスの腸バリア完全性の評価

Gut barrier integrity is a hallmark of intestinal health. While gut barrier integrity can be assessed using indirect markers such as the measurement of ...

分子動力学法を用いた脂質二重膜のシミュレーションと解析

Realistic Membrane Modeling Using Complex Lipid Mixtures in Simulation Studies

Lipids are structural building blocks of cell membranes; lipid species vary across cell organelles and across organisms. This variety results in different mechanical and structural properties in the membrane that directly impact the molecules and processes that occur at this interface. Lipid composition is dynamic and can serve to modulate cell signaling processes. Computational approaches are increasingly used to predict interactions between biomolecules and provide molecular insights to experimental observables. Molecular dynamics (MD) is a technique based on statistical mechanics that predicts the movement of atoms based on the forces that act on them. MD simulations can be used to characterize the interaction of biomolecules. Here, we briefly introduce the technique, outline practical steps for beginners who are interested in simulating lipid bilayers, demonstrate the protocol with beginner-friendly software, and discuss alternatives, challenges, and important considerations of the process. Particularly, we emphasize the relevance of using complex lipid mixtures to model a cell membrane of interest to capture the appropriate hydrophobic and mechanical environments in simulation. We also discuss some examples where membrane composition and properties modulate the interactions of bilayers with other biomolecules.

著者スポットライト:細胞膜生物物理学の進歩 - 実験的および計算的アプローチによる相互作用と課題の探求 

Membrane lipid diversity in structure and composition is an important contributor to cellular processes and can be a marker of disease. Molecular dynamics simulations allow us to study membranes and their interactions with biomolecules at atomistic resolution. Here, we provide a protocol to build, run, and analyze complex membrane systems.

シミュレーションスタディにおける複雑な脂質混合物を用いた現実的な膜モデリング

Lipids are structural building blocks of cell membranes; lipid species vary across cell organelles and across organisms. This variety results in different ...