この研究助成金から国立研究財団の韓国 (NRF) (Y m グラント号 2016R1A2B4008199 MSIP。 韓国政府によって資金を供給されるによって支えられましたH.) 厚生省と韓国の福祉 (許可番号: HI14C1324)。

すべてのプロシージャは、機関動物ケアおよび使用委員会の延世大学医科大学、韓国のガイドラインにしたがって運営しました。LysM 緑の蛍光蛋白質 (GFP; gfp/+) マウス8と CX3Cr1 GFP (gfp/+) 生体イメージングのための 8-12 週齢9マウスが使用されました。すべての手術器具はオートクレーブと 70% アルコールで消毒.
1. カスタム設計されたマウス肝イメージング ツールのサポート室
<ol><li>マウス肝イメージング室の設計<ol>
<li>(図 1) をイメージング マウス肝臓の次のサイズの部屋を作る: 内側のチャンバー (120 mm 長さ × 幅 160 mm × 30 mm 高さ)、外側室内 (100 mm 長さ × 幅 100 mm × 24 mm)、ボルト (Ф 3 × 30 mm 高さ)、ナット (Ф 4 mm)、と翼形ナット (Ф 4 mm)。</li>
		<li>硬化エージェントとエラストマーの比が 1:9 の基本とのシリコーンの 120 g の合計を測定します。</li>
		<li>液状シリコーン混合物を 250 mL の三角フラスコに入れて、よく混ぜてね。フラスコを乾燥器に入れて、ポンプを真空下で約 1 時間の空気の泡を削除します。</li>
		<li>カスタム設計されたマウスの商工会議所の高さ 10 mm ほどに液状シリコーン ミックスを注ぐそれをさせ (図 1 a) の日のカップルのための治療法。</li>
	</ol>
</li>
	<li>カバー スライドとベッドを固定レバーの準備<ol>
<li>金属製のフレームでは、シリコーン接着剤を使用してガラスを添付します。1 日 (図 1 b) で乾燥させます。</li>
		<li>水浸漬レンズ用蒸留水を効果的に保持するために半永久的な水ブロック構造が不可欠です。1 mm のシリコーン接着剤を丸めを行い、一晩それを強化します。</li>
		<li>硬化剤とエラストマー ベースのミックスし、それを注ぐ高さの約 8-10 mm。 実行 6 ウェル プレートに硬化プロセス 1.1.2-1.1.4 で説明したよう。</li>
	</ol>
</li>
</ol>2. 組合の準備
<ol><li>1 × リン酸緩衝生理食塩水 (PBS) 性腸炎サルモネラ血清型から LPS 粉末を希釈するための準備します。(1 × PBS: 137 mM 2.7 mM KCl 塩化ナトリウム 10 mM Na2HPO4、1.8 mM KH2PO4)。</li>
	<li>37 ° c の水浴の PBS を予熱して慎重に LPS 粉ボトル (25 mg) を開きます。</li>
	<li>ピペットで移しなさい援助を使用して、滅菌 PBS (5 mL) を注ぎ LPS パウダー ボトルと優しくかき混ぜるまたは在庫ソリューションを準備するソリューションを中断します。 注: LPS 粉末を液状、過酷なボルテックス泡が発生できます。1.5 mL チューブの因数を作るときに損失を最小限にするために泡を避けます。</li>
	<li>− 20 ° C、LPS 原液を格納し、注射用 1 mg/mL 組合ソリューションに希釈します。</li>
	<li>1 mg/mL の LPS 溶液 (20 mg/kg) を 1 mL の注射器を使用して非麻酔下マウスの腹膜に注入します。対照動物の PBS の同じボリュームを挿入します。</li>
	<li>十分な炎症性応答を 2 時間待つ。</li>
</ol>3. 血管を染色するテキサス赤デキストランの準備
<ol><li>2 つの 15 mL の円錐管と滅菌 PBS の 10 mL を準備します。</li>
	<li>テキサス赤デキストラン粉 (25 mg) 3 mL の PBS を希釈し、よく混ぜます。空 15 mL の円錐管に混合物を転送します。</li>
	<li>デキストランの混合物 (3 mL) を含む 15 mL の円錐管に残り PBS (7 mL) を転送します。</li>
	<li>0.45 μ m のシリンジ フィルターを 10 mL の注射器の先端に接続します。シリンジ フィルターが針を置き換える必要があります。</li>
	<li>デキストランの混合物 (10 mL) をフィルター処理します。こぼれることを避けるために均等に押し、注射器。</li>
	<li>光ブロック 1.5 mL チューブにデキストラン溶液 2.5 mg の 500 μ L を分注、− 20 ° C で保存解凍後、4 ° C で保存します。</li>
</ol>4. 手術のプロセス
<ol><li>手術テーブルと、手術前に 70% アルコールを使用してすべての機器を消毒します。肝切除のためのツールを設定し、37 ° c (図 2 a、 B) 加熱プレートを調整します。</li>
	<li>麻酔、腹腔チレタミン/チレタミンの 30 mg/kg とキシラジンの 10 mg/kg、マウスを注入します。これらのソリューションは、混合し、同時射出のため PBS で希釈できます。マウスのつま先を軽く押す anesthetization を確認します。 反射がない場合次の手順に進みます。</li>
	<li>マウスの網膜の乾燥を防ぐために眼軟膏の少量を適用します。</li>
	<li>脱毛クリーム (図 2) を使用して腹部周辺の体毛を削除します。</li>
	<li>尾静脈またはインスリン注射器 (図 2 D) を使用してレトロな軌道注入を介してテキサス赤デキストラン溶液 (2.5 mg/mL の濃度) の 200 μ L を注入します。</li>
	<li>マーカーで右弓下の領域をマークします。剣状突起から肋骨の終わりに線描する必要があります。鉗子、はさみ (図 2 e, F) で表皮をカットします。</li>
	<li>マイクロ剪刀、鉗子を用いた腹腔内を開き、慎重に肝臓を公開します。左葉外側綿棒 (図 2, H) を使用して展開する必要があります。</li>
	<li>肝臓の乾燥を防ぐために腹腔内に 500 μ L の滅菌 PBS を注ぐ。</li>
	<li>カスタム設計されたチャンバーで右側臥位でマウスを置き、シリコン ベッドに組織接着剤の少量を適用します。その後、綿棒 (図 2 i) を用いた肝慎重に取り付けます。 メモ: 肝臓の組織は非常にもろい、機械的に引っ張らないで、肝を。優しく、綿棒でそれをロールアウトします。</li>
	<li>数秒後は、500 μ L の肝臓から乾燥を防ぐため滅菌 PBS で再び肝臓を濡れています。肝臓に金属製のフレームを置き、ナット (図 2 j) でしっかりと固定します。</li>
</ol>5 2 光子顕微鏡を用いた肝臓の生体イメージング
<ol><li>880 nm、緑、セットし禅ソフトウェア (カールツァイス) レーザーをオンに、波長を設定するを実行し、赤のチャンネル。蛍光強度 (図 3 a) によるとレーザー パワーを調整します。排出フィルター情報のとおりです: グリーン/Alexa488、500-550 nm、赤/Alexa 555、575 610 nm。</li>
	<li>イメージングの段階でチャンバーを配置し、水浸対物レンズ カバー ガラスに近い X 20 を配置します。</li>
	<li>その体温 (図 3 b) を維持するためにマウスの体の下でシリコン ラバー ヒーターを修正します。</li>
	<li>金属製のフレームに 500 μ L の蒸留水をドロップ、肝臓 (図 3) に近い対物レンズを引き出します。</li>
	<li>イメージングの領域の深さを調べるし、フォーカスを調整します。</li>
	<li>イメージングを実施します。</li>
	<li>長期用チレタミン/チレタミンとキシラジンの混合物の半分線量イメージング中にそれぞれの時間を挿入します。この段階で、37 ° C でマウスの体温を維持することが不可欠です。常に目にマウスに anesthetization 時間とすべてのマウスは異なりますので。 注: このメソッドは手術とイメージング後回復に適していません。我々 は倫理的手術後すぐにマウスを犠牲します。</li>
	<li>安定した anesthetization の 30 分毎のペダルの反射神経をチェックします。</li>
</ol>6. データの分析
<ol><li>データ分析ソフトウェア Volocity を開き、ソフトウェアにライブラリ内の生データをインポートします。</li>
	<li>状況に合わせて、スナップショット画像からノイズを除去します。 注: 滑らかな画像を得るためには、'罰金または '中央' のフィルターを使用します。さらに、暗いまたはぼやけた画像を明確にするのに 'コントラスト強調' 機能を使用します。</li>
	<li>スナップショット画像を JPEG または TIFF ファイルと AVI ファイルの映画の 100%、輸出に編集した項目に適合します。</li>
</ol>

<ol>
	<li>Girard, J. R., 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=Fuels,+hormones,+and+liver+metabolism+at+term+and+during+the+early+postnatal+period+in+the+rat.">Fuels, hormones, and liver metabolism at term and during the early postnatal period in the rat.</a> J Clin Invest. 52 (12), 3190-3200 (1973).</li><li>Marques, P. E., 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=Imaging+liver+biology+in+vivo+using+conventional+confocal+microscopy.">Imaging liver biology in vivo using conventional confocal microscopy.</a> Nat Protoc. 10 (2), 258-268 (2015).</li><li>Dasari, S., Weber, P., Makhloufi, C., Lopez, E., Forestier, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+Microscopy+Imaging+of+the+Liver+following+Leishmania+Infection:+An+Assessment+of+Hepatic+Hemodynamics.">Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics.</a> J Vis Exp. (101), e52303 (2015).</li><li>Honda, M., 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=Intravital+imaging+of+neutrophil+recruitment+in+hepatic+ischemia-reperfusion+injury+in+mice.">Intravital imaging of neutrophil recruitment in hepatic ischemia-reperfusion injury in mice.</a> Transplantation. 95 (4), 551-558 (2013).</li><li>Jenne, C. N., Wong, C. H., Petri, B., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+spinning-disk+confocal+microscopy+for+the+intravital+analysis+of+platelet+dynamics+in+response+to+systemic+and+local+inflammation.">The use of spinning-disk confocal microscopy for the intravital analysis of platelet dynamics in response to systemic and local inflammation.</a> PLoS One. 6 (9), e25109 (2011).</li><li>Ritsma, L., 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=Intravital+microscopy+through+an+abdominal+imaging+window+reveals+a+pre-micrometastasis+stage+during+liver+metastasis.">Intravital microscopy through an abdominal imaging window reveals a pre-micrometastasis stage during liver metastasis.</a> Sci Transl Med. 4 (158), 158ra145 (2012).</li><li>Sierro, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Liver+Capsular+Network+of+Monocyte-Derived+Macrophages+Restricts+Hepatic+Dissemination+of+Intraperitoneal+Bacteria+by+Neutrophil+Recruitment.">A Liver Capsular Network of Monocyte-Derived Macrophages Restricts Hepatic Dissemination of Intraperitoneal Bacteria by Neutrophil Recruitment.</a> Immunity. 47 (2), 374-388 (2017).</li><li>Faust, N., Varas, F., Kelly, L. M., Heck, S., Graf, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insertion+of+enhanced+green+fluorescent+protein+into+the+lysozyme+gene+creates+mice+with+green+fluorescent+granulocytes+and+macrophages.">Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages.</a> Blood. 96 (2), 719-726 (2000).</li><li>Jung, S., 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=Analysis+of+fractalkine+receptor+CX(3)CR1+function+by+targeted+deletion+and+green+fluorescent+protein+reporter+gene+insertion.">Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion.</a> Mol Cell Biol. 20 (11), 4106-4114 (2000).</li><li>Heymann, F., 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=Long+term+intravital+multiphoton+microscopy+imaging+of+immune+cells+in+healthy+and+diseased+liver+using+CXCR6.Gfp+reporter+mice.">Long term intravital multiphoton microscopy imaging of immune cells in healthy and diseased liver using CXCR6.Gfp reporter mice.</a> J Vis Exp. (97), (2015).</li></ol>

著者は競合する金銭的な利益を宣言しません。

肝臓は多くのグループ3,10, その機能の重要性のために積極的に研究されています。例えば、ヘイマンらは agarose を使用して繊細な手法を提案します。ただし、このメソッドは、高精度なことが判明した、アガロースの設定時間がかかります。ただし、私たちの方法論ですべての手順が実施される他の交絡因子を最小限に抑え、30 分以内。ハートビ?...

肝臓は、哺乳類の主要な代謝器官それはエネルギー、ホルモンおよび解毒1の司令塔として機能します。その重要性のため研究者は炎症時に肝臓の変化を明らかにする多くの in vitroとin vivoの実験を行った。肝2からのライブ画像をキャプチャする生体顕微鏡を使用されています。確かに、イメージングの方法をされている様々 な肝臓は2,3,4,5,6を導入しました。しかしより簡易な外科的アプローチを開発、標的臓器や免疫細胞の安定化を達成するために、我々 は生体イメージングのための彼らの努力を最小限に抑えるための研究者を導くことができる単純なプロトコルを設計しました。
本研究では、安定的かつ新規イメージング室と出血や可能な限りの感染を最小限に抑えるために使用できる手術手技を紹介しました。肝臓は 2 時間以上そのまま残ったことを確かめた。この方法では、我々 は正常に LCMs7と敗血症性条件下における好中球の形態を識別されます。以来、このメソッドは、手術中に震えていると予期せぬ炎症など物理的・生物的交絡要因を最小化、このメソッドを使用して応答で肝臓の免疫細胞の急性反応を観察することができると見込んでください。試薬には、LP が好きです。

<table border="0" cellpadding="0" cellspacing="0" width="1004">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Custom designed chamber</td>
			<td style="width:239px;">Live Cell Instruments</td>
			<td style="width:272px;"></td>
			<td style="width:233px;">Custom-designed size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Metal cover slide</td>
			<td style="width:239px;">Live Cell Instruments</td>
			<td style="width:272px;"></td>
			<td style="width:233px;">Custom-designed size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Cover glass</td>
			<td style="width:239px;">Electron Microscopy Sciences</td>
			<td style="width:272px;">#72204-03</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Sylgard 184 Silicone elastomer kit</td>
			<td style="width:239px;">Dow Corning</td>
			<td style="width:272px;"></td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Erlenmeyer flask</td>
			<td style="width:239px;">DURAN</td>
			<td style="width:272px;">21 216 36</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Cell culture plate (Flat type)</td>
			<td style="width:239px;">HYUNDAI Micro Co.</td>
			<td style="width:272px;">H31006</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Desiccator</td>
			<td style="width:239px;">ADARSH</td>
			<td style="width:272px;">55204</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">High Q BOND SE 5 mL</td>
			<td style="width:239px;">BJM LAB</td>
			<td style="width:272px;"></td>
			<td style="width:233px;">To make semi-permanent dam</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Flexible Silicone Rubber Heaters</td>
			<td style="width:239px;">Omega</td>
			<td style="width:272px;">SRFR/SRFG SERIES, M1250/0800</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">DC power supply</td>
			<td style="width:239px;">Toyotech</td>
			<td style="width:272px;">DP30-03A</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Phosphate-buffered saline</td>
			<td style="width:239px;"></td>
			<td style="width:272px;"></td>
			<td style="width:233px;">Home-made</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:260px;">Lipopolysaccharides (LPS) from Salmonella enteria serotype enteritidis</td>
			<td style="width:239px;">Sigma-Aldrich</td>
			<td style="width:272px;">L6011</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Texas Red Dextran</td>
			<td style="width:239px;">Thermo Fisher Scientific</td>
			<td style="width:272px;">D1830</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:260px;">15 mL High-clarity polypropylene 
			conical tube</td>
			<td style="width:239px;">FALCON</td>
			<td style="width:272px;">14-959-49B</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:260px;">0.45 &#956;m filter (Minisart Syringe Filter)</td>
			<td style="width:239px;">Sartorius</td>
			<td style="width:272px;">16555k</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">1.5ml Micro Tube, Amber</td>
			<td style="width:239px;">Axygen</td>
			<td style="width:272px;">MCT-150-X</td>
			<td style="width:233px;">For light-blocking</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">1 mL syringe</td>
			<td style="width:239px;">Korea Vaccine</td>
			<td style="width:272px;">KV-S01</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">3 mL syringe</td>
			<td style="width:239px;">Korea Vaccine</td>
			<td style="width:272px;">KV-S03</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">10 mL syringe</td>
			<td style="width:239px;">Korea Vaccine</td>
			<td style="width:272px;">KV-S10</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">0.3 mL insulin syringe</td>
			<td style="width:239px;">Becton Dickinson</td>
			<td style="width:272px;">324900</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Hair removal cream 100 mL</td>
			<td style="width:239px;">Body natur</td>
			<td style="width:272px;"></td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Cotton swab</td>
			<td style="width:239px;">ABDI</td>
			<td style="width:272px;"></td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Zoletil 50 5 mL</td>
			<td style="width:239px;">Virbac</td>
			<td style="width:272px;"></td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Rompun 10 mL</td>
			<td style="width:239px;">Bayer</td>
			<td style="width:272px;"></td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Heating plate</td>
			<td style="width:239px;">Live Cell Instruments</td>
			<td style="width:272px;">PH-S-10</td>
			<td style="width:233px;">Custom-designed size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">DC controller</td>
			<td style="width:239px;">Live Cell Instruments</td>
			<td style="width:272px;">CU-301</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Microdessection Scissors</td>
			<td style="width:239px;">Harvard Apparatus</td>
			<td style="width:272px;">72-5418</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Scissors</td>
			<td style="width:239px;">Roboz</td>
			<td style="width:272px;">RS-5882</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Forceps</td>
			<td style="width:239px;">Roboz</td>
			<td style="width:272px;">RS-5137</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Vet bond</td>
			<td style="width:239px;">3M</td>
			<td style="width:272px;">1469SB</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">ZEN software (Black Edition)</td>
			<td style="width:239px;">Carl Zeiss</td>
			<td style="width:272px;"></td>
			<td style="width:233px;">Program for LSM 7 MP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">LSM 7 MP</td>
			<td style="width:239px;">Carl Zeiss</td>
			<td style="width:272px;"></td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Volocity</td>
			<td style="width:239px;">PerkinElmer</td>
			<td style="width:272px;"></td>
			<td style="width:233px;">Analysis program</td>
		</tr>
	</tbody>
</table>

two-photon intravital imaging of leukocytes during the immune response in lipopolysaccharide-treated mouse liver

敗血症は組織と臓器障害を引き起こす可能性が重度の感染症の種類の損傷。敗血症に関連する死亡率および罹患率率が非常に高く、ない直接の治療または臓器関連のメカニズムをリアルタイムで詳細に調べた。肝臓は毒素と人間の体内で感染症を管理する主要臓器であります。ここで、肝被膜マクロファージ (LCMs) 好中球などの免疫細胞の運動性を追跡するためにエンドトキシンの誘導後マウス肝臓の生体イメージングを実行する目的とします。したがって、露出低侵襲手術と肝臓の手術法を考案しました。リポ多糖 (LPS)、一般的なエンドトキシンを腹腔内に注入したマウス。露出と LPS 投与する LysM 緑の蛍光蛋白質 (GFP) でマウスの肝臓、その好中球がわかった募集の生体イメージングのための外科的手法を使用してマウスの肝臓はリン酸バッファーのそれと比べると増加しました。生理食塩液投与の肝。LPS 処理後の好中球数は時間と大幅に増加。また、CX3Cr1 GFP マウスを使用して、我々 は正常に、LCMs と呼ばれる肝マクロファージを可視化。したがって、生体内で免疫の移動を制御する新しい試薬の有効性を調べるためには、運動性および肝臓における好中球および LCMs の形態を決定することができによる臓器障害や組織損傷の治療の効果を識別するために敗血症における白血球の活性化。

シンプルで非常に安定した画像処理室が考案されました。チャンバーの下部は、マウス本体と臓器のすべりを防止するシリコンで覆われていた。また、シリコンは弾力性の適切な量があるのでそれはカバーのスライド (図 1 a, C) の圧力による損傷が予想されるを防ぐことができます。第二に、組織の安全な接着剤 (ボンド獣医) は、?...

Watch this Scientific Journal Video about Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver at JoVE.com

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

最小の侵入と生きたマウス肝臓の二光子励起イメージングのための新しい外科プロトコルを設立しました。この手法は、リポポリサッカライド誘起ブタエンドトキシン肝臓の微細構造を確認できた。肝の白血球の遊走に各種の試薬処理の有効性を決定するこのメソッドが利用されることを期待しています。

リポ多糖投与マウス肝臓における免疫応答時に白血球の二光子励起生体イメージング

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...

two-photon-intravital-imaging-leukocytes-during-immune-response

Research

JoVE Journal

Immunology and Infection

10.1K ビュー.  Yonsei University College of Medicine. 最小の侵入と生きたマウス肝臓の二光子励起イメージングのための新しい外科プロトコルを設立しました。この手法は、リポポリサッカライド誘起ブタエンドトキシン肝臓の微細構造を確認できた。肝の白血球の遊走に各種の試薬処理の有効性を決定するこのメソッドが利用されることを期待しています。

ビデオ: Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

の寄生蜂の紹介<em&gt;ショウジョウバエ</em&gt;と抗寄生虫免疫応答

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

免疫・感染学

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 ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

マウス膝窩リンパ節の生体内イメージング

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

免疫応答やスリープ中の定量的測定<em&gt;ショウジョウバエ</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

マウスマクロファージにおける先天性免疫応答を調査するために、RNA干渉を使用

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

西ナイルウイルス変異株の感染に対するMyD88非介在細胞性免疫応答のインビトロ分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various biological events in real time and in live animals. This technology has greatly advanced our understanding of physiological processes and pathogen-mediated phenomena in specific organs.
In this study, IVM is applied to the mouse liver and protocols are designed to image in vivo the circulatory system of the liver and measure red blood cell (RBC) velocity in individual hepatic vessels. To visualize the different vessel subtypes that characterize the hepatic organ and perform blood flow speed measurements, C57Bl/6 mice are intravenously injected with a fluorescent plasma reagent that labels the liver-associated vasculature. IVM enables in vivo, real time, measurement of RBC velocity in a specific vessel of interest. Establishing this methodology will make it possible to investigate liver hemodynamics under physiological and pathological conditions. Ultimately, this imaging-based methodology will be important for studying the influence of L. donovani infection&#160;on hepatic hemodynamics.
This method can be applied to other infectious models and mouse organs and might be further extended to pre-clinical testing of a drug&#8217;s effect on inflammation by quantifying its effect on blood flow.

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

以下の肝臓の生体顕微鏡イメージング<em&gt;リーシュマニア</em&gt;感染症：肝血行動態の評価

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various ...

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, ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

のゼブラフィッシュ幼生モデルにおける先天性免疫応答の非侵襲的イメージング<em&gt;ストレプトコッカスイニエ</em&gt;感染

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Cell Membrane Repair Assay Using a Two-photon Laser Microscope

Numerous pathophysiological insults can cause damage to cell membranes and, when coupled with innate defects in cell membrane repair or integrity, can result in disease. Understanding the underlying molecular mechanisms surrounding cell membrane repair is, therefore, an important objective to the development of novel therapeutic strategies for diseases associated with dysfunctional cell membrane dynamics. Many in vitro and in vivo studies aimed at understanding cell membrane resealing in various disease contexts utilize two-photon laser ablation as a standard for determining functional outcomes following experimental treatments. In this assay, cell membranes are subjected to wounding with a two-photon laser, which causes the cell membrane to rupture and fluorescent dye to infiltrate the cell. The intensity of fluorescence within the cell can then be monitored to quantify the cell's ability to reseal itself. There are several alternative methods for assessing cell membrane response to injury, as well as great variation in the two-photon laser wounding approach itself, therefore, a single, unified model of cell wounding would beneficially serve to decrease the variation between these methodologies. In this article, we outline a simple two-photon laser wounding protocol for assessing cell membrane repair in vitro in both healthy and dysferlinopathy patient fibroblast cells transfected with or without a full-length dysferlin plasmid.

Cell membrane wounding via two-photon laser is a widely used method for assessing membrane resealing ability and can be applied to multiple cell types. Here, we describe a protocol for in vitro live-imaging of membrane resealing in dysferlinopathy patient cells following two-photon laser ablation.

2 光子レーザー顕微鏡を用いた細胞膜修復法

Numerous pathophysiological insults can cause damage to cell membranes and, when coupled with innate defects in cell membrane repair or integrity, can ...