著者は、フライブルク大学（CSに）医学部からドイツ学術振興SI-1281/4-1とBIOSにからの補助金によって支援を認める。

1。病原性抗体の作製注意：抗体は一晩0.1MグリシンpHは2.5から3（溶出バッファー）（ON）に格納されるべきではないとして精製と抗体の濃度は、同じ日に行われるべきである。 IgGのアフィニティー精製：免疫ウサギ血清25mlを使用します。 <ol><li> 4でONウサギ血清を融解℃で、20℃で1xPBS（バッファを実行している）と、それを1:1で混合し、10分間1260 xgでそれを遠心血清脂肪酸である場合には必要に応じて、ろ紙でろ過工程は、遠心分離後に含まれている場合があります。 </li><li>一方、G行列は1XPBSの10ベッドボリューム（ベッドボリューム=マトリックス容積（）バッファを実行している）でカラムを充填したタンパク質を洗う。 </li><li>カラムに希釈し、濾過した血清を適用して、室温（RT）でロッキングプラットフォーム上で1時間インキュベートする。 </li><li>フロースルー（FT）は50ミリリットルの小瓶チューブ内を収集します。濃度までそれを捨てないでくださいと精製IgGの力価が知られている。 </li><li> 1XPBSの5ベッドボリュームで行列を洗い、その間に溶出したIgG画分を収集するために50mlのFalconチューブを準備する。ピペット1ミリリットルのpH中和バッファー：各チューブの1MトリスpH 10（そうでなければ、収集する溶出液の量に対する1時20分TRIS緩衝液の割合）。 </li><li>カラムに0.1MグリシンpHは2.5から3と50 mlの画分（s）を収集してください。100〜150ミリリットルの溶出バッファーを適用する。 2〜3倍管を裏返して、pHを測定し、pHが7.2から7.4に達するまで中和バッファーを追加します。 </li><li> IgGの溶出液数滴を収集し、エタロンは280nmでODを測定するよう溶出緩衝液を用いて。 ODは&lt;0.1ストップ収集している場合。 </li><li>プロテインG行列を再生成し、製造元の指示に従って保管してください。 </li></ol>アミコン管と限外ろ過による濃縮： <ol><li> 15-20分を遠心分離することによって1XPBSとアミコンウルトラ（15ミリリットル/ 30KDa）限外濾過管を洗う4℃、3220×gでアミコンからFTを捨ててください。 </li><li> 3220 xgで25から30分間溶出したIgG画分、遠心15mlでAmiconsと4を埋める℃、遠心分離時間は常に溶出液のタンパク質含有量に依存します。 </li><li> FTを捨て、あなたは残っていない溶出画分を持っていないまで限外ろ過を繰り返す。 </li><li>遠心分離により1XPBS 25から30分間で、広範囲に、2回濃縮したIgGを洗ってください。 </li><li> 1XPBSの1,500-2,000最終量100μlのアミコンフィルターから &quot;スティッキー&quot;、黄色がかった抗体溶液を収集します。 </li><li>分光光度計や光度計を用いてIgG調製物の濃度を測定します。 コンク。 （mg / ml）でA = A（280 nm）の×希釈率/ 1,4 </li><li>製造元の指示に従ってAmiconsを洗って保管してください。 </li></ol> 2。マウスにVII型コラーゲン特異的IgGの注射実際の実験手順を開始する前に、次のことを確認した実験プロトコールが書き込まれ、すべての材料は、実験のために用意されている。 <ol><li>病気の得点、ELISA法、蛍光抗体法（IF）の分析とミエロペルオキシダーゼ（MPO）アッセイ4のデータシートを準備します。 </li><li>血液や臓器、cryomoldsならびに組織像処理や埋カセット用チューブにラベルする。 </li><li>抗体溶液を、新しく調製または株式から解凍し、その反応性（IF顕微鏡および/またはELISAによる力価）に関して特徴付けられるべきである。滅菌濾過抗体溶液を注入するために必要な線量は250から1000μlの間で変化するようにPBSで希釈する必要があります。あなたは全体の実験を実行するための十分なIgGを持っていることを確認してください。 </li><li>ヘパリン20単位/ mlおよび8.7 mg / mlのケタミン/麻酔用1.3 mg / mlのキシラジン混合物：新鮮な抗凝固剤を準備します。マウスを犠牲にすると準備3.7％ホルムアルデヒドでチューブラベルを貼っている。 </li><li> （メス、はさみ、細かい点とcurv手術器具を滅菌エドforcepsの複数形）と注射器（インスリン注射器、1と2 mLシリンジ）、針、デジタルカメラや余分なメモリカードを準備します。 </li></ol> 注：ケタミン/キシラジンまたはイソフルラン麻酔マウス上のすべての手順を実行したマウスは素早く回復としてイソフルランは、毎日の肌チェックのための好ましい麻酔のオプションです。しかし、写真を撮るときに、87 mg / kgのケタミンおよび13 mg / kgをキシラジンは、通常、皮下注射する。安楽死のためにケタミン/キシラジン投与量は130 mg / kgのケタミンおよび20 mg / kgをキシラジンに増加しています。 <ol><li>彼らの一般的な健康状態、皮膚、毛皮の外観のために既にマークの動物を確認し、それらを比較検討すると、（常に同じ耳に固執する）は、その耳の厚さを測定します。 </li><li>観測値と臨床評価シート内の変更を登録します。 </li><li>抗凝固剤の同じボリュームを含むシリンジで尾静脈20から30μL（2〜3滴）の血液を採取します。 </li><li>毛皮を消毒し、80％エタノールを使用した肌。 </li><li> 。皮下インスリン注射器を使用して、バックに抗体溶液を注入する（特定のサイト（例えば、耳）の場合のみの小さなボリューム（10-50μL）を注入することが可能です。） </li></ol>注射は、一日おきに4回繰り返す必要があります。 BALB / cおよび約20グラムの体重のC57BL6マウスは3-4日最大750μg/ gの体重/注射自己抗体に400から500の最初の投与後に疾患の開発を開始。 実験を終了する場合： <ol><li>皮下に130 mg / kgおよび20 mg / kgのケタミン/キシラジンを注入することにより、深い麻酔下にマウスを置き、後部大静脈からそれらを血を抜くと心臓を切り取ってください。 </li><li>皮膚などの器官/組織サンプル収集：尻尾、耳、血液や食道を、以前にラベル付けされ、PBSまたは3.7％ホルムアルデヒドは充填チューブに入れます。 </li></ol>に戻るとき実験室： <ol><li> 1200×gで、独立したプラズマに室温で10分間で血液を遠心分離し、適切にラベルしたチューブにプラズマを転送し、それに応じてそれらを格納します。 </li><li>皮膚が正常cryomolds、ラベルを使用して最適なカット温度培地で生検し、-80℃で保管して埋め込む℃に</li><li>ラベルされた埋め込み型の組織学のために意味の組織サンプルを配置し、パラフィン包埋までの3.7％ホルムアルデヒドに保管してください。 </li></ol> 3。疾患の重症度の臨床評価マウスは、皮膚や粘膜の病変を毎日検討すべきであるとの知見は、適切なフォームを使用して記録されるべきである。早期に安楽死のための基準は、5の疾患スコア（ 表1）のカウントは、よりその20体表面の％と3日間連続で全体重の5〜10％の体重減少に影響を与え、皮膚病変です。 <ol><li>その腹の上にマウスを置きます。右側で、すなわち頭部領域で開始耳と内側にだけでなく、外側に水疱、びらん、地殻を確認してください。左耳と同じように続ける。 </li><li>左右の目が脱毛症、紅斑の徴候を制御し、びらん及び/又はクラストしています。 </li><li>おでこと鼻領域は鼻、唇や口の粘膜の腹の部分を続けると、次の通ってブラッシングされています。 </li><li>上向きに動く、足から始めて右前脚を調べ、左前脚に切り替えます。左右の後ろ足と同じ手順。 </li><li>湾曲した細かい点鉗子で首と背中エリアの毛皮を介してブラシ。 </li><li>その背中の上でマウスの電源を入れ、腹側だけでなく、手足の腹側以前と同じようにチェックしてください。 </li><li>尾を確認してください。 </li><li>マウスは疾患を発症関連の身体の部分の写真を撮る。注目は写真の質と量に支払わなければならない。 </li><li>臨床疾患スコアを計算。 </li></ol> 4。皮膚および血漿サンプルの分析<ol><li> 、店の準備をし、および/または計画に従って収集したすべての組織を処理します。 </li><li>ウサギ抗体とマウス補体系成分のIFを検出するための凍結組織切片を切り取ります。 </li><li>凍結組織および/または器官抽出物の異なるタンパク質と酵素含量（MPOアッセイ）を分析します。 </li><li>セクションパラフィン包埋組織と組織の損傷の程度を視覚化するためにH＆Eで染色。 </li><li> ELISA、イムノブロットにより血漿を分析します。 </li></ol>

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実験動物への自己抗体の受動的な転送は、その病原性7、-12を実証するための主要なアプローチです。この方法により得られた動物モデルは、自己免疫13の間接的な証拠であることに加えて、病原性機構の遠心性の相の調査を可能にします。後天性表皮水疱症（EBA）の膨れ抗体誘導性顆粒球依存皮膚の受動的伝達モデルは、自己免疫性皮膚疾患における組織損傷のメカ?...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> 注釈 </td></tr><tr><td>組換え抗原</td><td></td><td></td><td>マウスVII型コラーゲンの組換え形態をコードするプラスミドは、対応する著者から提供されています。 </td></tr><tr><td>免疫ウサギ血清</td><td> <a href="http://www.eurogentec.com/" target="_blank">www.eurogentec.com</a> </td><td></td><td>我々は、抗原200μgの、2週間隔で3回で免疫されたニュージーランドホワイトウサギを持っていた。このために、我々はEurogentec、SA、ベルギーのサービスを使用している。 </td></tr><tr><td>プロテインGアガロース</td><td>ロシュ·ダイアグノスティックス</td><td> 11243233001 </td><td></td></tr><tr><td> Balb / cマウス</td><td>チャールズ·リバー·ラボラトリーズ</td><td></td><td></TD&gt; </tr><tr><td> OCTコンパウンド、ティッシュテック</td><td>サクラファインテック</td><td> 4583 </td><td> 10月、最適な切削温度</td></tr><tr><td> Cryomold標準</td><td>サクラファインテック</td><td> 4557 </td><td> 25ミリメートル×20ミリメートル×5ミリメートル</td></tr><tr><td> Cryomold中間</td><td>サクラファインテック</td><td> 4566 </td><td> 15ミリメートル×15ミリメートル×5ミリメートル</td></tr><tr><td>ユニリンクEinbettkassette </td><td> R. Langenbrinck </td><td> 09から0503 </td><td>組織学処理や埋カセット</td></tr><tr><td> SPEZIAL-Tatowierfarbeシュワルツ</td><td> H. Hauptnerund＆リチャードHerberholz社＆Co KG </td><td> 71492000 </td><td>ペーストを刺青</td></tr><tr><td> Tatowierzange TZ1 </td><td> EBECO E.ベッカー＆カンパニー社</td><td></td><td>刺青装置</td></tr><tr><td>ヘパリン</td><td>カール·ロート社＆Co。 </td><td> 7692.1 </td><td></td></tr><tr><td> 37％ホルムアルデヒド</td><td>カール·ロート社＆Co。 </td><td> 7386 </td><td></td></tr><tr><td>塩酸ケタミン</td><td>シグマアルドリッチケミー</td><td> K2753-1G </td><td></td></tr><tr><td>キシラジン塩酸塩</td><td>シグマアルドリッチケミー</td><td> X1251-1G </td><td></td></tr><tr><td>滅菌PBS </td><td> Biochrom </td><td> L182-50 </td><td></td></tr><tr><td>デジタルカメラ</td><td>ニコン</td><td> COOLPIX 5400 </td><td></td></tr><tr><td>シリンジフィルターユニット0.45μmのを牽引</td><td>ミリポア</td><td></td><td></td></tr><tr><td>キャリパー</td><td>ミツトヨ7309 </td><td> 1667338 </td><td>ファーネル（ディストリビュータ） </td></tr><tr><td>病気のスコアシート</td><td></td><td></td><td>柵の例SED </td></tr></tbody></table>

granulocyte-dependent autoantibody-induced skin blistering

自己免疫現象は、健康な個体で発生しますが、自己寛容が失敗したときに、自己免疫応答が特定の病状になることがあります。によるWitebskyのは、自己免疫疾患などの診断の基準のいずれかを仮定すると、自己抗体の受動的伝達によって、実験動物における疾患の再生です。後天性表皮水疱症（EBA）、皮膚や粘膜の原型の臓器特異的自己免疫疾患のために、いくつかの実験モデルは、最近設立された。 VII型コラーゲンの200アミノ酸（aa 757から967）のストレッチに対してIgG抗体を精製し、我々の現在の仕事で説明する動物モデルで猛烈な表現型だけでなく、人間に特徴的な病理組織および免疫病理学的特徴を再現したマウスに繰り返し注入されEBA 1。本格的な広範な疾患は、通常、最初の注射後5-6日見られ、疾患の程度は、投与されたCの線量と相関しているollagen VII-特異的IgG。実験EBAにおける組織損傷（ブリスター形成）-4組織結合自己抗体2によって顆粒球の動員および活性化に依存している。モデルは唯一の遠心性自己免疫応答のT細胞非依存位相を再現したがって、このモデルでは、自己免疫組織損傷に関与する顆粒球依存性炎症経路の解剖を可能にします。また、その値は、in vivoで自己抗体のブリスター誘導可能性を示すとEBA 1,3、-6における水疱形成のメカニズムを調査研究の数によって強調される。最後に、このモデルでは、大幅に自己抗体によって誘発される疾患における新たな抗炎症治療薬の開発を容易にするでしょう。全体的に、EBAの受動転送動物モデルがアクセス可能で有益な疾患モデルであると研究者はEBAの病因ではないだけを分析するのではなく、基本的な生物学的にも臨床的に答えるのに役立ちます本質的な自己免疫の質問。

マウスでは、本格的な疾患における抗原特異的抗体結果の受身伝達、臨床組織学的および免疫病理学的なレベルで人間EBA似ている。水疱、痂皮、びらん、脱毛症は、耳、鼻、足、脚、背中、マウスの目の周りに開発しています。病気の最初の臨床症状は、おそらく耳および/または頭部領域に表示されます。特定ウサギIgG、およびマウス補体C3の沈着は、IF病変部周囲の皮膚の凍結切片で真皮?...

Watch this Scientific Journal Video about Granulocyte-dependent Autoantibody-induced Skin Blistering at JoVE.com

Granulocyte-dependent Autoantibody-induced Skin Blistering

VII型コラーゲンの200アミノ酸（aa 757から967）のストレッチに対してIgG抗体を精製し、我々の現在の仕事で説明する動物モデルで猛烈な表現型だけでなく、人間に特徴的な病理組織および免疫病理学的特徴を再現したマウスに繰り返し注入され後天性表皮水疱症（EBA）<sup&gt; 1</sup&gt;。

顆粒球に依存する自己抗体誘発皮膚水疱形成

Autoimmune phenomena occur in healthy individuals, but when self-tolerance fails, the autoimmune response may result in specific pathology. According to ...

granulocyte-dependent-autoantibody-induced-skin-blistering

Research

JoVE Journal

Immunology and Infection

10.4K ビュー.  University of Freiburg. VII型コラーゲンの200アミノ酸（aa 757から967）のストレッチに対してIgG抗体を精製し、我々の現在の仕事で説明する動物モデルで猛烈な表現型だけでなく、人間に特徴的な病理組織および免疫病理学的特徴を再現したマウスに繰り返し注入され後天性表皮水疱症（EBA） 1。

ビデオ: Granulocyte-dependent Autoantibody-induced Skin Blistering

Cutaneous Leishmaniasis in the Dorsal Skin of Hamsters: a Useful Model for the Screening of Antileishmanial Drugs

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of lesion size is a hard procedure and animals show difficulty to eat, breathe and move because of the lesion. In order to optimize the hamster model for cutaneous leishmaniasis, young adult male and female golden hamsters (Mesocricetus auratus) were injected intradermally at the dorsal skin with 1 to 1.5 x l07 promastigotes of Leishmania species and progression of subsequent lesions were evaluated for up to 16 weeks post infection. The golden hamster was selected because it is considered the adequate bio-model to evaluate drugs against Leishmania as they are susceptible to infection by different species. Cutaneous infection of hamsters results in chronic but controlled lesions, and a clinical evolution with signs similar to those observed in humans. Therefore, the establishment of the extent of infection by measuring the size of the lesion according to the area of indurations and ulcers is feasible. This approach has proven its versatility and easy management during inoculation, follow up and characterization of typical lesions (ulcers), application of treatments through different ways and obtaining of clinical samples after different treatments. By using this method the quality of animal life regarding locomotion, search for food and water, play and social activities is also preserved.

Optimization of the experimental hamster model for cutaneous leishmaniasis by intradermal injection of Leishmania promastigotes at the dorsal skin. This approach is useful during inoculation, follow-up, characterization of lesions, application of treatments and obtaining of clinical samples. Locomotion, search for food and water, play and social activities are preserved.

ハムスターの背部皮膚における皮膚リーシュマニア症：Antileishmanial薬剤のスクリーニングのための有用なモデル

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of ...

免疫・感染学

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

合成脂質二重膜上でHIV-1エンベロープ誘発性ウイルス学的シナプスとシグナリングのイメージング

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

マウスにおける同種のCD4 T細胞応答を研究する尾皮膚の移植

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

表現型分析のためのヒト皮膚からリンパ球単離と<em&gt; ex vivoで</em&gt;細胞培養

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and epidermis home a variety of immune cells in both homeostatic and inflammatory conditions. Tools to obtain skin cell release for cytofluorimetric analyses are, therefore, very useful in order to study the complex network of immune cells residing in the skin and their response to microbial stimuli. Here, we describe an efficient methodology for the digestion of mouse skin to rapidly and efficiently obtain single-cell suspensions. This protocol allows maintenance of maximum cell viability without compromising surface antigen expression. We also describe how to take and digest skin samples from different anatomical locations, such as the ear, trunk, tail, and footpad. The obtained suspensions are then stained and analyzed by flow cytometry to discriminate between different leukocyte populations.

The skin is home to a complex immune cell network. We describe an efficient methodology for the digestion of mouse skin, from different parts of the animal's body, in order to obtain a single-cell suspension and analyze the different leukocyte populations resident in the skin by flow cytometry.

別のマウス皮膚の領域から細胞蛍光分析のための単一細胞懸濁液の調製

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

酵素消化および勾配分離を使用したマウス皮膚から浸潤白血球の単離

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

弱毒で皮内免疫後のマウスの皮膚及び排出リンパノードから骨髄性細胞の単離<em&gt;マラリア原虫</em&gt;スポロゾイト

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ability to switch cells from one pathway to another is highly attractive as a therapeutic target. We designed a decoy peptide IRF5D with a molecular modeling software for designing small molecules and peptides.
IRF5D inhibited IRF5, reduced alterations in extracellular matrix, and improved endothelial vasodilation in the tight-skin mouse (Tsk/+). The Kd of IRF5D for recombinant IRF5 is 3.72 &plusmn; 0.74 x 10-6 M as determined by binding experiments using biolayer interferometry experiments. Endothelial cells (EC) proliferation and apoptosis were unchanged using increasing concentrations of IRF5D (0 to 100 &#181;g/mL, 24 h). Tsk/+ mice were treated with IRF5D (1 mg/kg/d subcutaneously, 21 d). IRF5 and ICAM expressions were decreased after IRF5D treatment. Endothelial function was improved as assessed by vasodilation of facialis arteries from Tsk/+ mice treated with IRF5D compared to Tsk/+ mice without IRF5D treatment. As a transcription factor, IRF5 traffics from the cytosol to the nucleus. Translocation was assessed by immunohistochemistry on cardiac myocytes cultured on the different cardiac extracellular matrices. IRF5D treatment of the Tsk/+ mouse resulted in a reduced number of IRF5 positive nuclei in comparison to the animals without IRF5D treatment (50 &#181;g/mL, 24 h). These findings demonstrate the important role that IRF5 plays in inflammation and fibrosis in Tsk/+ mice.

We describe the isolation of cardiac extracellular matrix from C57Bl/6J control mice, tight-skin mice, and tight-skin mice treated with the IRF5 inhibitory peptide. We also describe the vasodilation studies on the isolated vessels from C57Bl/6J, tight-skin mice and tight-skin mice treated with the IRF5 inhibitory peptide.

単離された血管の血管拡張およびタイト皮膚マウスの細胞外マトリックスの単離

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ...

Murine Full-thickness Skin Transplantation

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited application to humans, skin transplantation in mice has been widely employed for transplantation research. The procedure is easy to learn and perform, and it does not require delicate microsurgical techniques nor extensive training. Moreover, graft rejection in this model occurs in a very reproducible immunological reaction and is easily monitored by direct inspection and palpation. In addition, secondary skin transplantation with donor-matched or third-party skin grafts can be performed on more complex transplant models as an alternative and uncomplicated method to assess donor-specific tolerance. The complications are low and are in general limited to anesthesia overdose or respiratory distress after the procedure. Graft failure, on the other hand, occurs commonly as a result of poor preparation of the graft, incorrect positioning in the graft bed, or inappropriate placement of the bandage. In this article, we present a protocol for full-thickness skin transplantation in mice and describe the important steps necessary for a successful procedure.

Murine full-thickness skin transplantation is a well-established model to study rejection in an alloimmune setting. Here, we provide a tutorial of each step involved in performing a BALB/c--&#62;C57BL/6 full-thickness skin transplant.

マウス全層皮膚移植

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...

Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a fascinating approach to investigate immune modulation of cutaneous inflammation in mouse models of human diseases. Here we present a cost-effective protocol that delivered naked DNA in mouse skin and leads to transgene expression. The method is coined &#34;acufection&#34;, denoting acupuncture-mediated DNA transfection. To perform acufection, mouse skin was first infused with DNA in phosphate-buffered saline (PBS) and then pricked lightly with a bundle of acupuncture needles to facilitate the absorption of DNA and transfection into cells. The plasmid DNA is presumably taken up by the keratinocyte and dendritic cells (DCs) in the skin and expressed into protein. Mechanical prick with the needles per se did not cause skin damage or induce keratinocyte activation. The expression of the transfected genes was detected in the skin at both transcriptional and translational levels following acufection for 2 days and maintained up to 7 days. The primary goal for the development of this acufection method was to investigate a previously undefined isoform of IL-15. Using this method, an alternatively spliced IL-15 isoform with partially deleted exon 7 (IL-15&#916;E7) was expressed in the skin and subsequently treated with a Toll-like receptor 7 (TLR7) agonist, imiquimod (IMQ), to induce inflammation. Acufection-delivered IL-15&#916;E7 in skin suppressed keratinocyte proliferation, epidermal thickness and neutrophil recruitment in IMQ-induced cutaneous inflammation. With increasing interest in identifying the regulatory mechanisms of cutaneous inflammation, the protocol described here provides a cost effective and versatile alternative to the gene gun system or microseeding for DNA delivery in vivo. It may potentially allow discovery of the function of a novel gene in the skin or for investigating new treatment for cutaneous diseases.

Development of an Economical DNA Delivery System by &quot;Acufection&quot; and its Application to Skin Research

This protocol is a cost-effective alternative for expressing naked plasmid DNA in mouse skin. The overall goal of the protocol is to deliver immune-related genes into skin tissue to delineate the functional role of a specific gene in cutaneous inflammation.

「Acufection」と肌の研究への応用により、経済的なDNAデリバリーシステムの開発

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a ...