研究所臨床 Hemostaseology と輸血ドナー血液を提供するために感謝します。カルメン Hässig と優秀な技術的なヘルプのコーラ Hoxha。LifeAct mEGFP 構造を生成するありがとう修正 pMAX ベクトルのイェンス レッティグ (ザールラント州大学)、ローランド Wedlich-日ソールドナー (ミュンスター大学) 元 LifeAct ルビー構築のため、キリスト教のユンカー (ザールラント州大学)。このプロジェクトは、Sonderforschungsbereich 1027 (プロジェクト A2 ベクレル) と 894 (プロジェクト A1 か) によって賄われていた。DFG によって資金が供給された光シート顕微鏡 (GZ: INST 256/4 19 1 FUGG)。

ひと由来の材料 (ヒトの血液ドナーからの白血球減少システム室) と本研究の遂行の研究が倫理委員会 (16.4.2015 (84/15; から宣言によって承認されて教授博士レッティグ Stürmer))、対応するガイドラインに従います。
1. 中和コラーゲン溶液の調製 (500 μ L)
<ol><li>細胞文化のフードの下で滅菌 1.5 mL チューブに冷やしたコラーゲン原液 (10.4 mg/mL) の 400 μ L を転送します。徐々 に 50 μ L の冷蔵チルド コラーゲン原液の 400 μ L に 10 x PBS (pH 7.0 7.3) を追加します。チューブにそっと並べてソリューションをミックスします。 注: セル文化フードの下でパート 1 のすべてのステップを行ってください。</li>
	<li>1.1 から 500 μ L のコラーゲン溶液に 0.1 M NaOH の 8 μ L を追加します。7.2 7.6 に pH を調整します。PH テスト ストリップを使用 (pH 範囲: 6-10) 混合物の pH 値を確認します。 注: ボリュームは、コラーゲンの異なるバッチの異なる場合があります。NaOH 溶液が空気の泡を避けるためにゆっくりと混合します。混合物は、コラーゲンのゲル化を避けるために氷の上に保管すべき。</li>
	<li>追加 2 μ L 滅菌 ddH2O 500 μ L に最終巻をするはよく混合し、さらに使用するまで氷の上または 4 ° C でこのコラーゲン溶液 (8.32 mg/mL) を格納します。 注: この条件の下で中和されたコラーゲン使用できます 24 時間因数は空気の泡を避けるために推奨されません。</li>
</ol>2. 毛細血管を使用して光シート蛍光顕微鏡用試料
<ol><li>蛍光前述目的主な蛍光染料15または蛍光タンパク質11で関心の生きているセルにラベルを付けます。</li>
	<li>細胞文化のフードの下で滅菌 1.5 mL チューブに 1 × 106セルを転送します。200 x g で 8 分間破棄でチューブに上清を遠心し、培養液 200 μ L でペレットを再懸濁します。 注: 特に細胞傷害性 T リンパ球 (CTL) とナチュラル キラー (NK) 細胞のための人間の免疫細胞の移動の可視化のためには、5 × 106セル/mL の細胞密度の使用をお勧めします。</li>
	<li>1.3 から中和されたコラーゲン溶液の 85.9 μ L を追加します。2.2 からセル中断。2.5 mg/mL のコラーゲン濃度に到達する適切にミックスします。ボンネットの氷にセル/コラーゲン ミックスを残します。 注: N mg/mL、コラーゲンの必要な濃度と、仮定のボリューム中和 (200 μ L 細胞懸濁液) のコラーゲン溶液 200 × N =/(8.32-N)</li>
	<li>次に、一致するプランジャーを毛細血管に入れ (内径 〜 1 mm) ピストンが毛細血管から 1 mm になるまで。培地 (図 1 a) に浸漬によってプランジャーを濡れています。 注: この手順は、セル/コラーゲン ミックスにキャピラリーを浸したときに空気の泡を防ぐために助けることができます。キャピラリーとピストンが無菌であることはありません。</li>
	<li>2.3 からセル/コラーゲン ミックスにキャピラリーを浸し。10 〜 20 ミリメートル (図 1 b) にプランジャーをゆっくり引き出します。残りのコラーゲン溶液を削除する 70% エタノール スプレーで湿らせたペーパー タオルで毛細血管の外側の壁を拭いてください。</li>
	<li>マウント 5 mL チューブの内壁に粘土のモデリング、毛管、キャピラリー (図 1) の端にセル/コラーゲン ミックスを押してください。</li>
	<li>5% CO2コラーゲン重合の 1 h の 37 ° C で毛細血管を保ちます。</li>
	<li>5 mL チューブに培 (約 1-2 mL) を追加します。(図 1) の中にぶら下がっているコラーゲンの約 3/4 媒体に、重合コラーゲン棒を慎重に押して。</li>
	<li>5% CO2培地でコラーゲン ロッドを平衡に別の 30 分の 37 ° C で、このような毛細血管を保ちます。 注: その後、コラーゲン棒引っ張ることができる毛管と培養にさらに使用する前にさらに。</li>
</ol>3. 画像集録光シート顕微鏡を用いた
<ol><li>アセンブリの製造元の指示に従って試料室。</li>
	<li>孵化とサンプル室 (ライブセル イメージングのみ) の 37 ° C に加熱する顕微鏡を入れます。</li>
	<li>試料室にキャピラリーを置きイメージ獲得のため関心のある分野を検索するサンプルを探します。</li>
	<li>対応する laser(s) をアクティブにします。次の設定: レーザー パワー、露光時間、z スタック、および生きているセルイメージ投射の時間間隔の z スタック ・開始・終了位置のステップ サイズ。 メモ: たとえば、図 2の例をイメージしましたすべての 40 s 1 μ m のステップ サイズの 37 ° C で 6 時間 (総厚: 538 μ m)。レーザー出力が 30 さん x、y 方向のピクセル サイズの露光時間で 1 %0.23 μ m。</li>
	<li>画像の取り込みを開始します。</li>
</ol>4. 自動追跡の分析
<ol><li>ファイル コンバーターを開き、ソフトウェアのファイル形式 (*.ims) に変換する画像ファイルを選択して[ファイルの追加] をクリックします。参照をクリックし、変換後のファイルを保存するフォルダーを選択します。すべてを開始をクリックします。</li>
	<li>解析ソフトウェアを開きます。凌駕をクリックします。ファイルに移動し、クリックしてオープン、分析対象とする画像ファイルを選択します。 注: ファイルのサイズが大きい場合この手順は時間をかかることがあります。1 テラバイト (TB) を超えるファイルには推奨されません単一の実験プロセスとしてこのような大きなファイルは高い計算能力を要求します。</li>
	<li>新しいスポットを追加クリックします。チェック ボックスを最終的に全体の画像を処理します。[次へ] をクリックします。</li>
	<li>X の座標を入力し、y (ピクセル) に関心の領域を定義します。(時間および z 位置) を分析するフレーム数を入力します。[次へ] をクリックします。</li>
	<li>ソース チャンネルのドロップ ダウン リストで追跡するオブジェクトが含まれるターゲット チャネルを選択します。(Μ m) で推定される xy の直径を入力します。[次へ] をクリックします。 注: xy の推定直径は追跡するオブジェクトの x と y 次元の平均粒径です。</li>
	<li>[品質] をクリックします。細胞 (オブジェクト) が含まれているでする必要があります、しきい値を設定します。[次へ] をクリックします。</li>
	<li>必要なアルゴリズム (自己回帰運動をお勧めします) を選択します。最大距離を入力 (20 μ mをお勧めします) と最大ギャップ サイズ (3 または 2 を推奨)。プロセス全体の最後に画像をクリックします。 注:最大距離と最大ギャップ サイズは、トラックを分割する 2 つのしきい値です。具体的には、2 つの連続的なフレームで同じオブジェクト間の距離が最大距離を超えたらそれ以降のフレームでこのオブジェクトとみなされます新しいオブジェクト。買収の実行中に同じオブジェクト可能性がありますいくつかのフレームの消え、再び現れます。この場合、最大ギャップ サイズ内でこのオブジェクトが表示されるときにだけは、同じオブジェクトとしては考えられます。</li>
	<li>フィルターの種類をクリックし、不要なトラックを除外するオプションを選択します。 注: この手順は省略できます。</li>
	<li>次をクリックし、[完了] をクリックします。 注: このステップ性能に応じて日まで時間がかかります。</li>
	<li>トラックの編集をクリックし、正しいドリフトを選択します。(並進ドリフトをお勧めします) 適切なアルゴリズムを選択します。結果のデータセットのサイズを選択 (現在のサイズに等しい新しいサイズが推奨)。[Ok]をクリックします。 注: この手順はのみ画像集録中にコラーゲンが漂流したときに必要な。</li>
	<li>[統計情報] をクリックし、リスト表示されている統計値の構成を選択します。関心のオプション エクスポート (例えば座標、速度など) を確認します。[Ok]をクリックします。</li>
	<li>[すべての統計をファイルにエクスポート] をクリックし、ファイル名を入力します。</li>
</ol>5. 固定と蛍光抗体法コラーゲンの細胞の染色
<ol><li>化学のフードの下で 5 mL チューブに 4% パラホルムアルデヒド (PFA、PBS で) の 1,000 μ L を転送します。 注: PFA は、部屋の温度にバランスする必要があります。</li>
	<li>2.9 から重合コラーゲンと毛細血管を浸しなさい。PFA のソリューションに (の ~ 5 mm) と (図 1で示すように)、粘土のモデリングと 5 mL チューブの内壁に毛細血管をマウントします。</li>
	<li>PFA ソリューション (図 1) にぶら下がっているコラーゲン ロッドの半分までプランジャーを軽く押します。20 分間室温でチューブを維持します。</li>
	<li>キャピラリー内コラーゲン ロッドを得るにプランジャーを引き戻します。毛細血管を取り出して、PFA を破棄します。</li>
	<li>新鮮なチューブにキャピラリーをマウントし、1 mL の PBS を追加します。毛細血管が PBS に没頭していることを確認します。</li>
	<li>ソリューションにぶら下がっているコラーゲン ロッドの半分までプランジャーを軽く押します。粘土のモデリングと内壁に毛細血管をマウントします。</li>
	<li>5 分間室温でチューブを維持します。</li>
	<li>5.4 を繰り返します。-別の 2 回の 5.7。</li>
	<li>キャピラリー内コラーゲン ロッドを得るにプランジャーを引き戻します。PBS を破棄します。チューブに 1-2 mL のブロック/透過バッファー (PBS + 1 %bsa + 0.1% 非イオン性界面活性剤) を転送し、5.6 を繰り返します。 注: セカンダリ Ab で育てられた動物の 5% 血清による BSA (1%) を置き換えることができます。</li>
	<li>30-60 分間室温でチューブを維持します。</li>
	<li>キャピラリー内コラーゲン ロッドを得るにプランジャーを引き戻します。透過バッファーを破棄します。ブロック/透過バッファーの一次抗体の 200-500 μ L を転送し、ソリューションにロッドを追放。</li>
	<li>1 時間室温でチューブを維持します。</li>
	<li>5.4 で説明したように PBST (PBS + 0.1-% 非イオン性界面活性剤) で 3 回コラーゲン棒を洗ってください。-5.8。</li>
	<li>室温で 1 時間ブロッキング/透過バッファーで二次抗体のロッドを孵化させなさい。光からそれを維持します。</li>
	<li>5.4 で説明したように PBS で 3 回コラーゲン棒を洗ってください。-5.8。</li>
	<li>キャピラリー内コラーゲン ロッドを得るにプランジャーを引き戻します。イメージングまで PBS のサンプルを保ちます。</li>
	<li>3 で説明したように、サンプルをスキャンします。</li>
</ol>

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+sectioning+deep+inside+live+embryos+by+selective+plane+illumination+microscopy.">Optical sectioning deep inside live embryos by selective plane illumination microscopy.</a> Science. 305 (5686), 1007-1009 (2004).</li><li>Verveer, P. 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=High-resolution+three-dimensional+imaging+of+large+specimens+with+light+sheet-based+microscopy.">High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy.</a> Nat Methods. 4 (4), 311-313 (2007).</li><li>Truong, T. V., Supatto, W., Koos, D. S., Choi, J. M., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+and+fast+live+imaging+with+two-photon+scanned+light-sheet+microscopy.">Deep and fast live imaging with two-photon scanned light-sheet microscopy.</a> Nat Methods. 8 (9), 757-760 (2011).</li><li>Haycock, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+cell+culture:+a+review+of+current+approaches+and+techniques.">3D cell culture: a review of current approaches and techniques.</a> Methods Mol Biol. 695, 1-15 (2011).</li><li>Mestas, J., Hughes, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+mice+and+not+men:+differences+between+mouse+and+human+immunology.">Of mice and not men: differences between mouse and human immunology.</a> J Immunol. 172 (5), 2731-2738 (2004).</li></ol>

著者は金融または商業上の利害を宣言しません。

ほとんどの in vitroアッセイは 2 D 画面で、たとえば細胞培養プレート、シャーレや coverslips、生体内で細胞、免疫細胞特に経験ほとんど 3次元微小環境に対し行われます。出現の証拠は、2 D と 3 D のシナリオ17で免疫細胞の移行パターンが異なることを示しています。さらに、腫瘍細胞の発現も 2 D と 3 D 培養組織18,19<su...

移行についてほとんど知識細胞に由来する 2 D 実験1,2,3、通常ガラスまたは文化/イメージングのプラスチック表面で実施された料理。しかし、生理学的なシナリオを必要とするほとんどのケースで 3 D 微小環境、細胞外マトリックス (ECM) が決定的な役割を果たしています。ECM は、適切な細胞の形態を維持するために 3 D 構造エッセンシャルを提供するだけでなく、生存信号または多く細胞4、5の最適な機能の方向性の手がかりを提供しています。したがって、3 D 環境が良い細胞機能および生理学的なコンテキストを反映してより良い環境での動作を識別するために必要です。
人間の体内では、ほとんど細胞免疫細胞特には 3 D シナリオの下でその機能を発揮します。たとえば、活性化 T 細胞は標的細胞を探して組織をパトロール、ナイーブ T 細胞はリンパ節の検索中に移行モードと機械に対応する細胞外適応している同種の抗原提示の細胞に移行環境3,6,7。3次元コラーゲンゲルは、よく確立され、よ特徴付けられた 3 D の細胞培養システム8,9,10として広く使用されています。我々 の以前の仕事は、一次ヒトリンパ高度なモバイルが 0.25% コラーゲン ベースの行列11約 4.8 μ m/分の平均速度で移行を示しています。細胞骨格の再編は、細胞移行12のキーの役割を果たしています。細胞外シグナルが曲とリンパ球のみのシングル モードの移行は適用されませんまだ場所、微小環境、サイトカイン、走化性の勾配に応じて特定の移行動作を切り替えることができますを示しています証拠を蓄積、別の方法3の回遊行動。
免疫細胞機能や動作、たとえば、移行、突出部形成または小胞輸送を確実に分析するには、高速かつ信頼性の高い方法で比較的大きな 3 D ボリューム内の画像を取得することができるため大きな利点です。3 D イメージングのため (1 つの平面照明顕微鏡とも呼ばれる) 最近開発された光シート顕微鏡技術は、満足のいく解決13,14を提供しています。買収、イメージング、中にサンプルを照らす照明板が薄く静的が生成されます。これで、焦点面大面積は、平面を細胞に影響を与えずに同時に照らされることができます。この機能により、大幅に削減漂白と photocytotoxicity 高速です。本稿では、光シート顕微鏡を用いたプライマリひと免疫細胞を可視化する方法と 3 D のシナリオで移行を分析する方法について説明します。

<table>
	<tbody>
		<tr>
			<td>Fibricol, bovine collagen solution</td>
			<td>Advanced Biomatrix</td>
			<td>&#160;#5133-20ML</td>
			<td>Collagen matrix</td>
		</tr>
		<tr>
			<td>0.5 M NaOH Solution</td>
			<td>Merck</td>
			<td>1091381000</td>
			<td>for neutralizing Fibricol solution</td>
		</tr>
		<tr>
			<td>Ultra-Low melting agarose</td>
			<td>Affymetrix</td>
			<td>32821-10GM</td>
			<td>Sample preparation in low c[Col]</td>
		</tr>
		<tr>
			<td>Dynabeads Untouched Human CD8 T Cells Kit</td>
			<td>Thermo Fisher</td>
			<td>11348D</td>
			<td>Isolation of primary human CD8+ T cells from PBMC</td>
		</tr>
		<tr>
			<td>Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation</td>
			<td>Thermo Fisher</td>
			<td>11132D</td>
			<td>Activation of CTL populations</td>
		</tr>
		<tr>
			<td>Human recombinant interleukin-2</td>
			<td>Thermo Fisher</td>
			<td>PHC0023</td>
			<td>Stimulation of cultured CTL</td>
		</tr>
		<tr>
			<td>P3 Primary solution kit</td>
			<td>Lonza</td>
			<td>V4XP-30XX</td>
			<td>Transfection</td>
		</tr>
		<tr>
			<td>&#945;-PFN1 antibody, rabbit, IgG</td>
			<td>Abcam</td>
			<td>ab124904</td>
			<td>IF</td>
		</tr>
		<tr>
			<td>Alexa Fluor 633 Phalloidin</td>
			<td>Thermo Fisher</td>
			<td>A22284</td>
			<td>IF</td>
		</tr>
		<tr>
			<td>CellMask Orange Plasma membrane Stain</td>
			<td>Thermo Fisher</td>
			<td>C10045</td>
			<td>Fluorescent cell label</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma</td>
			<td>P1379-250mL</td>
			<td>IF</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Eurobio</td>
			<td>018774</td>
			<td>IF</td>
		</tr>
		<tr>
			<td>DPBS Dulbecco&#39;s phopsphate buffered saline</td>
			<td>Thermo Fisher</td>
			<td>14190250</td>
			<td>IF</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A9418-100G</td>
			<td>IF</td>
		</tr>
		<tr>
			<td>Goat &#945; Rabbit 568, IgG, rabbit</td>
			<td>Thermo Fisher</td>
			<td>A-11011</td>
			<td>IF</td>
		</tr>
		<tr>
			<td>Lightsheet Z.1 (Light-sheet microscopy)</td>
			<td>Zeiss</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture hood</td>
			<td>Thermo Fisher</td>
			<td>HeraSafe KS</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture incubator HERACell 150i&#160;</td>
			<td>Thermo Fisher</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5418 and 5452</td>
			<td>Eppendorf</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr>
			<td>Pippettes</td>
			<td>Eppendorf</td>
			<td>3123000039, 3123000020, 3123000063</td>
			<td></td>
		</tr>
		<tr>
			<td>Pippette tips</td>
			<td>VWR</td>
			<td>89079-444, 89079-436, 89079-452&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL tubes</td>
			<td>Sarstedt</td>
			<td>&#160;62.554.002</td>
			<td></td>
		</tr>
		<tr>
			<td>Capillaries 50 &#181;L</td>
			<td>VWR (Brand)</td>
			<td>613-3373</td>
			<td>Zeiss LSFM sample preparation</td>
		</tr>
		<tr>
			<td>Plunger for capillaries</td>
			<td>VWR (Brand)</td>
			<td>BRND701934</td>
			<td>&#34;Stamps with Teflon tip&#34; LSFM sample preparation</td>
		</tr>
		<tr>
			<td>MColorPhast pH stips</td>
			<td>Merck</td>
			<td>1095430001</td>
			<td>to test pH of neutralized Fibricol</td>
		</tr>
		<tr>
			<td>BD Plastipak 1mL syringes</td>
			<td>BD</td>
			<td>Z230723&#160;ALDRICH</td>
			<td>Alternative sample preparation</td>
		</tr>
		<tr>
			<td>Modeling clay (Hasbro Play-Doh A5417EU7)</td>
			<td>Play-Doh</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris file converter</td>
			<td>Bitplane</td>
			<td>available at http://www.bitplane.com&#160;</td>
			<td>Convert imaging files to Imaris file format</td>
		</tr>
		<tr>
			<td>Imaris 8.1.2 (MeasurementPro, Track, Vantage)</td>
			<td>Bitplane</td>
			<td>available at http://www.bitplane.com&#160;</td>
			<td>Analysis of 3D and 4D imaging data</td>
		</tr>
	</tbody>
</table>

light-sheet microscopy for three-dimensional visualization of human immune cells

生体内で、活性化、増殖、およびすべての免疫細胞の機能は、例えばリンパ節の組織の三次元 (3 D) 環境で発生します。最新では、ほとんどの生体外でシステムは細胞培養プレートか coverslips などの 2 次元 (2 D) 表面に依存します。生理学的な条件を最適な模倣の in vitro、単純な 3 D コラーゲン マトリックスを駆使します。コラーゲンは細胞外マトリックス (ECM) の主要なコンポーネントの 1 つと広く 3 D マトリックスを構成するために使用されています。3 D イメージングの (単一の平面照明顕微鏡とも呼ばれる) 最近開発された光シート顕微鏡技術は高い集録速度、大きな浸透深さ、低漂白、photocytotoxicity と紹介されています。さらに、光シート顕微鏡は長期計測のため特に有利です。ここで最適化されたプロトコルは、セットアップ、人間の免疫細胞、例えば主なひと細胞傷害性 T リンパ球 (CTL) を処理する方法を記述してナチュラル キラー (NK) 細胞の生細胞イメージングのための光シート顕微鏡用 3 D コラーゲン マトリックス ・固定サンプル。細胞遊走の画像取得と解析の手順が掲載されています。特定焦点は、重要なステップと試料調製及びデータ解析のための要因を強調表示に与えられます。このプロトコルは、3 D コラーゲン マトリックスの懸濁細胞の他のタイプのために用いることができるし、免疫細胞に制限されていません。

T 細胞の移行中に突出部形成は、アクチン依存である非常にダイナミックなプロセスです。主なヒト CTL の突起形成を視覚化するには、一過性11の前に、の前述の CTL におけるアクチン細胞骨格のラベルに mEGFP 融合タンパク質を transfected しました。トランスフェクション後、1 日、細胞は、コラーゲン マトリックスに埋め込まれていた。画像のスタッ...

Watch this Scientific Journal Video about Light-sheet Microscopy for Three-dimensional Visualization of Human Immune Cells at JoVE.com

Light-sheet Microscopy for Three-dimensional Visualization of Human Immune Cells

人間の免疫細胞の三次元可視化のための光シート顕微鏡

ここでは、光シート顕微鏡を用いた三次元 (3 D) コラーゲン マトリックスに埋め込まれた免疫細胞を可視化するためのプロトコルを提案する.このプロトコルはまた、3 D の細胞の移動を追跡する方法を詳しく説明します。3 D マトリックスの懸濁細胞の他のタイプのこのプロトコルを用いることができます。

In vivo, activation, proliferation, and function of immune cells all occur in a three-dimensional (3D) environment, for instance in lymph nodes or ...

light-sheet-microscopy-for-three-dimensional-visualization-human

Research

JoVE Journal

Immunology and Infection

8.0K ビュー.  Saarland University. ここでは、光シート顕微鏡を用いた三次元 (3 D) コラーゲン マトリックスに埋め込まれた免疫細胞を可視化するためのプロトコルを提案する.このプロトコルはまた、3 D の細胞の移動を追跡する方法を詳しく説明します。3 D マトリックスの懸濁細胞の他のタイプのこのプロトコルを用いることができます。

ビデオ: Light-sheet Microscopy for Three-dimensional Visualization of Human Immune Cells

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

マウス大動脈内の免疫細胞のフローサイトメトリー分析

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

免疫・感染学

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

ヒトのインビトロ</em免疫バランスの初期状態の認識のためのスクリーニングツールとして>抑制

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

2光子顕微鏡を用いてマウスの胸腺の生体内イメージング

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

脂肪組織の免疫細胞の単離

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion (STED) Nanoscopy

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic actin reorganization is critical to the function of NK cells and the formation of the IS. Imaging of F-actin at the synapse has traditionally utilized confocal microscopy, however the diffraction limit of light restricts resolution of fluorescence microscopy, including confocal, to approximately 200 nm. Recent advances in imaging technology have enabled the development of subdiffraction limited super-resolution imaging. In order to visualize F-actin architecture at the IS we recapitulate the NK cell cytotoxic synapse by adhering NK cells to activating receptor on glass. We then image proteins of interest using two-color stimulated emission depletion microscopy (STED). This results in &#60;80 nm resolution at the synapse. Herein we describe the steps of sample preparation and the acquisition of images using dual color STED nanoscopy to visualize F-actin at the NK IS. We also illustrate optimization of sample acquisition using Leica SP8 software and time-gated STED. Finally, we utilize Huygens software for post-processing deconvolution of images.

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion STED Nanoscopy

Here we illustrate the protocol for imaging by two-color STED nanoscopy the cytotoxic immune synapse of NK cells recapitulated on glass. Using this method we obtain sub-100 nm resolution of synapse proteins and the cytoskeleton.

デュアルカラータイム·ゲーティッド誘導放出枯渇（STED）ナノスによる免疫シナプスの可視化

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

CXCR6.Gfpレポーターマウスを使用して、健康で罹患肝臓における免疫細胞の長期生体内多光子顕微鏡イメージング

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

ヒト末梢血の免疫表現型分析のための抗体のカクテルを準備する半自動のアプローチ

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

Isolation and Identification of Extravascular Immune Cells of the Heart

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with functional compartmentalization both during steady state and during different forms of inflammation. Until recently, the study of immune cells in the heart required the use of microscopy or poorly developed digestion protocols, which provided enough sensitivity during severe inflammation but were unable to confidently identify small &#8212; but key &#8212; populations of cells during steady state. Here, we discuss a simple method combining enzymatic (collagenase, hyaluronidase and DNAse) and mechanical digestion of murine hearts preceded by intravascular administration of fluorescently-labelled antibodies to differentiate small but unavoidable intravascular cell contaminants. This method generates a suspension of isolated viable cells that can be analyzed by flow cytometry for identification, phenotyping and quantification, or further purified with fluorescence-activated cell sorting or magnetic bead separation for transcriptional analysis or in vitro studies. We include an example of a step-by-step flow cytometric analysis to differentiate the key macrophage and dendritic cell populations of the heart. For a medium sized experiment (10 hearts) the completion of the procedure requires 2&#8211;3 h.

This protocol presents a simple and efficient method to isolate, identify and quantify immune cells residing in the myocardium of mice during steady state or inflammation. The protocol combines enzymatic and mechanical digestion for the generation of a single cell suspension that can be further analyzed by flow cytometry.

心の血管外免疫細胞の分離と同定

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

単一細胞の蛍光顕微鏡による Efferocytosis の定量化

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be affected by cell shape. Bacterial cells are three-dimensional (3D) objects, although they are rarely treated as such. Most microscopy techniques result in two-dimensional (2D) images leading to the loss of data pertaining to the actual 3D cell shape and localization of proteins. Certain shape parameters, such as Gaussian curvature (the product of the two principal curvatures), can only be measured in 3D because 2D images do not measure both principal curvatures. Additionally, not all cells lie flat when mounting and 2D imaging of curved cells may not accurately represent the shapes of these cells. Accurately measuring protein localization in 3D can help determine the spatial regulation and function of proteins. A forward convolution technique has been developed that uses the blurring function of the microscope to reconstruct 3D cell shapes and to accurately localize proteins. Here, a protocol for preparing and mounting samples for live cell imaging of bacteria in 3D both to reconstruct an accurate cell shape and to localize proteins is described. The method is based on simple sample preparation, fluorescent image acquisition, and MATLAB-based image processing. Many high-quality fluorescent microscopes can be simply modified to take these measurements. These cell reconstructions are computationally intensive and access to high-throughput computational resources is recommended, although not necessary. This method has been successfully applied to multiple bacterial species and mutants, fluorescent imaging modalities, and microscope manufacturers.

This protocol explains how to prepare and mount bacterial samples for live three-dimensional imaging and how to reconstruct the three-dimensional shape of E. coli from those images.

正確な細胞表現と正確なタンパク質局在化のための細菌細胞の三次元イメージング

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be ...