この作品は、技術革新 (CFI) のアルバータ大学学部医学と歯学、友人のギャレット カミング研究椅子基金、HM のトゥーピン神経科学研究の椅子基金、筋ジストロフィー カナダ、カナダの財団によって支えられました。アルバータ州の高度な教育と技術 (AET)、カナダ保健研究 (機構)、ジェシーの旅 - 遺伝子と細胞療法、女性と子供の健康研究所 (WCHRI) のための基礎機構とアルバータ州革新健康ソリューション (AIHS).
博士スティーブン ラヴァルをありがとうフルレングス ジスフェリン プラスミドをご提供したいと思います。技術的助言の Katsuya Miyake 先生に感謝も申し上げます。

本研究で使用されるひと線維芽細胞は、医学部・歯学部アルバータ大学で人間の倫理委員会から承認を得て使用されました。
1. フルレングスの DYSF プラスミドを細胞のトランスフェクション
<ol>
	<li>成長媒体 - 36 mL ダルベッコ変更イーグル培地 (DMEM)、ウシ胎児血清 (FBS) の 4 mL、ペニシリン/ストレプトマイシン (P/S) - 37 ° C で CO2インキュベーターの 20 μ L の 40 mL を含む T225 フラスコ培養線維芽細胞 注: セルは約 70-80% の confluency に培養する必要があり、なって完全に合流するべきではないです。</li>
	<li>シードの細胞を準備をするには、40 ml のリン酸緩衝生理食塩水 (PBS) のセルを 2 回、リンス セルをデタッチする 0.05% トリプシンの 5 mL を追加します。37 ° c CO2インキュベーターのセルを返すし、5 分のセルを孵化させなさい。<ol>
		<li>セルは完全に独立した、トリプシン反応を停止する成長媒体の 40 mL を追加します。</li>
		<li>診断を使用してセルまたはデバイスを数える別のセルをカウントします。</li>
	</ol>
	</li>
	<li>35 mm コラーゲン被覆ガラス底皿に5セル/mL の 1 x 10 のセルの 2 mL をシードします。37 ° C で CO2インキュベーターで一晩細胞をインキュベートします。 注: セルする約 50-60% 合流次の日する必要があります。</li>
	<li>成長媒体を取り外して 2 ml の血清を奪われたメディア (FBS なし成長媒体)。</li>
	<li>フルレングス ジスフェリン プラスミド lipotransfection を使用してセルを transfect します。<ol>
		<li>1.5 mL チューブ (トランスフェクションするのに各料理の 1 つの管) の血清を奪われたメディアの 150 μ L を準備します。</li>
		<li>血清を奪われたメディアを含む各 1.5 mL チューブにトランスフェクション試薬の 3 μ L を追加します。それは室温で少なくとも 5 分間インキュベートします。</li>
		<li>別 1.5 mL チューブに 175 μ L の血清を奪われたメディアとプラスミド DNA の 3.5 μ g を追加します。 注: は、トランスフェクションするのに各追加料理の上記金額を乗算します。</li>
		<li>150 μ L の血清を奪われたメディアとトランスフェクション試薬の 150 μ L でチューブにプラスミド DNA を含むメディアを組み合わせます。室温で 20 分間インキュベートそれ。</li>
		<li>プラスミド、トランスフェクション試薬とメディアの 300 μ L を料理に均等に分散します。37 ° C で CO2インキュベーターで 24 h の料理を孵化させなさい</li>
	</ol>
	</li>
	<li>血清を含む新鮮な成長媒体でメディアを交換し、37 ° C で CO2インキュベーターでさらに 24 時間インキュベート</li>
</ol>
2 セルの 2 光子負傷試金のための準備
<ol>
	<li>ピペッティングでメディアを削除、タイロード溶液 1 mL と 1 回細胞を洗浄し、2.5 ミリメートル FM の 1 μ L を含む新鮮なタイロード溶液の 1 mL を追加 4 64 染料。 注: FM 4-64 の染料の最終濃度は 2.5 μ M です。</li>
	<li>カルシウムの欠乏した環境で細胞膜修復の評価が必要な場合は、1 ml の PBS のセルをすすいで 1 mM グリコールエーテルジアミン四酢酸を含む PBS の 1 つの mL を追加します。 注: PBS は、細胞が時間の経過を切断すると負傷のアッセイは、できるだけ早く完了する必要があります。</li>
</ol>
3. 2 光子レーザー負傷アッセイ
<ol>
	<li>倒立の共焦点顕微鏡と関連付けられたプログラム ソフトウェアを使用して、0.2 μ x 2 μ m ターゲットを作成し、細胞膜の端にそれを置き、ターゲット ラインと重なるし、細胞膜 (図 2) の方向に垂直であります。</li>
	<li>FM 色素信号を励起し、600-760 nm の検出範囲の設定 543 nm HeNe レーザーを使用します。GFP 信号 (図 4) をエキサイト 500 550 nm の検出範囲を設定し、488 nm アルゴン レーザーを使用します。<ol>
		<li>作成の 5 分の時系列連続画像スキャン、イメージ投射セルのすべての 5 s。</li>
		<li>膜病変を生成するには、820 nm、15% を使用してに設定 2 光子レーザーを用いた漂白剤細胞はレーザ 10 の反復処理で出力と時系列の初めの後細胞 25 s をブリーチします。</li>
	</ol>
	</li>
	<li>FM 4-64 色素蛍光を定量化するには、ソフトウェアを使用して、利益 (率 ROI) の 6 μ x 6 μ m 領域を描画し、負傷の場所と (図 3) のセル内に配置。 注: ソフトウェアで-このアプリケーションで「範囲インジケーター」関数を使用してピクセル過飽和のサンプリング エリアを避けるために、赤いピクセルは、過飽和ピクセルを表します。統計分析のための電子スプレッドシートに未加工の蛍光値をエクスポートします。</li>
	<li>(膜を傷つける前に縦長の平均蛍光値) バック グラウンド値を減算して各時点の相対的な蛍光値を計算し、t 増加額を割って、蛍光値 = 0 (図 5, 6).</li>
</ol>

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2 光子レーザーの細胞膜の負傷は、再シールする体外膜のダイナミクスを評価するため正確で汎用性の高い手法です。この記事で dysferlinopathy 患者細胞アッセイが負傷した二光子励起レーザーを使用しての能力を再シールする細胞膜を決定するプロトコルについて述べる。Dysferlinopathy 患者の細胞は細胞膜が再シールに欠陥が他の研究者による調査結果に一貫しているし、筋肉間の原動?...

真核細胞の細胞膜は、細胞の内か細胞外の環境を定義し、細胞の恒常性と細胞の生存を維持するために不可欠ですタンパク質がちりばめられたリン脂質の二重層で構成されます。機械的又は化学的侮辱から生じる細胞膜損傷、様々 な哺乳類の細胞のタイプで平凡、骨格を含む、心筋、胃、肺細胞は1,2,3,4。毎日生理機能の結果としての傷害、に加えては、環境の侮辱、細菌毒素、虚血再灌流5によって細胞膜を破損ことができます。細胞膜の破裂を再シールに失敗につながる細胞外 Ca2 +- - その他の潜在的に有害細胞コンポーネントと共に無秩序な流入、細胞へ細胞になることができますすぐに下流の信号カスケードをトリガー死1,4,5,6。
日には、細胞膜修復のためいくつかの提案されたモデルがずっとあります。サイズや膜破裂の性質に応じて異なる修復メカニズムが起動できます。たとえば、側方流動や目詰まり小さな混乱を修復するタンパク質が細胞膜を利用することが示唆された (&#60; 1 nm)。側面の融合モデルを提案する膜が破裂するがジスフェリン含む横募集を通して出される急速に膜7、蛋白質モデルの目詰まり小さな穿孔がタンパク質集計を出されることを示唆しています。(大抵アネキシン)8します。 逆に、大きい膜病変を引き起こす Ca2 +-依存、ジスフェリンを介した小胞融合し修復パッチの形成。修理パッチ モデルでセルに急速な Ca2 +流入トリガー形複合体や細胞内小胞の融合と共に「パッチ」複数のタンパク質 (ジスフェリン、アネキシン、mitsugumin-53、EDH 蛋白質) の募集やリソソーム膜のサイトで4,9,10、11,12に損傷を与えます。これらのモデルは必ずしも相互に排他的ではありませんし、膜の修復を容易にするコンサートで動作可能性がありますに注意してくださいすることが重要です。細胞膜の損傷を正しく再シールに失敗した筋ジストロフィー (dysferlinopathy - 三好ミオパチー、肢帯型筋ジストロフィーのタイプ IIB、前方脛骨発症型ミオパチーなど) を含む複数の病気の状態に関連付けられています。13、心筋症の14、および Chediak-東症候群 (CHS)15。
その適切な細胞膜の完全性と演劇を再シール健康と病気、そのような重要な役割細胞膜修復の分子機構の解明理解有益となる新しい治療戦略の検索で。したがって、速度を監視し、細胞膜修復の能力を評価する適切な実験技術を保有する必要は。細胞膜修復をモデル化するためいくつかの培養方法を設計されています。1 つの戦略には、機械的損傷、細胞をこするピペット/手術/刃またはセル16のガラスビーズの寝返りによってを通じて促進することができますが含まれます。ただし、機械的損傷のこのタイプはより大きな病変を生成し、細胞損傷、内や文化の間の変化の高度を作成します。
膜の傷を生成する別の方法は、二光子励起顕微鏡法によるレーザーアブレーションです。単一光子励起を採用し従来のレーザー共焦点顕微鏡と対照をなして 2 光子レーザーは同時に17の高エネルギー電子の励起を容易にするのに 2 つの長波長、低エネルギーの光子を使用します。この非線形プロセス結果励起焦点面でのみ、全体の光路17 (図 1) にはないです。これは、削減励起ボリューム イメージングの生きているセル18,19時に光損傷を最小限に抑えることです。研究者は、細胞膜とモニター膜は蛍光染料を使用し、細胞膜が破裂し、reseals としての蛍光強度の変化を観察することによってリアルタイムで再封止の正確な病変を生成することが従って。
このアプローチは、 in vitroとin vivoが負傷した膜を研究に使用されました、複数セルのタイプ20,21。たとえば、膜は、線維芽細胞および dysferlinopathy この技術22,23を行った患者由来細胞の欠陥を修復します。また、マウスから隔離された単一の筋線維は、修復パッチ形成13,24,25を監視する使用されました。9単一の筋線維の細胞膜修復中の蛍光付けられた蛋白質の動きを観察することも。さらに、ゼブラフィッシュ胚における二光子励起レーザーが負傷した後, 修復のプロセスは、26リアルタイム体内で観察できます。
この記事で私たちは能力を再封止膜の定量化を目的として様々 な細胞タイプにこの方法論を適用できますが、2 光子レーザーの負傷を使用して線維芽細胞細胞膜修復の動態を評価するための方法論を概説します。生体外で。FM4 64 セルに孵化するこの方法では、すぐに否定的に連結され、蛍光を発する脂溶性、細胞不浸透性染料の帯電膜病変から細胞に入ると細胞質内リン脂質 (図 2&#38;3). 色素蛍光膜病変に隣接の定量化により細胞膜自体を封印するためにかかる時間を監視するため。このメソッドの有用性を実証するため細胞膜修復の救助を評価するのに GFP 共役フルレングス ジスフェリン (DYSF) プラスミドをトランスフェクトした dysferlinopathy 患者線維芽を使用します。

<table border="0" cellpadding="0" cellspacing="0" width="600">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:71px;">Thermo Fisher</td>
			<td style="width:119px;">11320033</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Fetal Bovine Serum</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">F1051</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:71px;">Thermo Fisher</td>
			<td style="width:119px;">15140122</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Trypsin-EDTA (0.05%), phenol red</td>
			<td style="width:71px;">Thermo Fisher</td>
			<td style="width:119px;">25300062</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">35mm collagen-coated glass-bottom dishes</td>
			<td style="width:71px;">MatTek</td>
			<td style="width:119px;">P53GCOL-1.5-14-C</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Serum-deprived media</td>
			<td style="width:71px;">Thermo Fisher</td>
			<td style="width:119px;">31985070</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Transfection reagent</td>
			<td style="width:71px;">Thermo Fisher</td>
			<td style="width:119px;">15338100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">FM 4-64 Dye</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">T13320</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Tyrode&#8217;s Salts Solution</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">T2397</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Confocal laser scanning microscope</td>
			<td style="width:71px;">Carl Zeiss</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Chameleon Two-photon laser</td>
			<td style="width:71px;">Coherent</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

cell membrane repair assay using a two-photon laser microscope

数多くの病態生理学的侮辱は細胞膜に損傷を与えることができるし、細胞膜修復または整合性の生来欠陥と相まって病で発生する可能性が。周囲の細胞膜修復基盤となる分子機構の解明は、したがって、機能不全の細胞膜の動態と関連する疾患の新規治療戦略の開発に重要な目的。細胞膜を再シールするさまざまな疾患のコンテキストで理解することを目的とした多くのin vitroとin vivoの研究は、次の実験的治療機能の結果を決定するための標準として 2 光子レーザー焼灼を利用します。この分析では、細胞膜が破裂し、細胞に侵入する蛍光色素細胞膜をおこす二光子励起レーザーと負傷にさらされます。セル内での蛍光の強度は、自体を再シールする細胞の能力を定量化する監視できます。アプローチ自体が負傷した二光子レーザーで大きな変化と同様、傷害、細胞膜の応答を評価するためのいくつかの方法があります、したがって、セルが負傷したの単一の統一されたモデルは有益減少になるだろう、これらの方法のバリエーション。この記事では、細胞膜修復体外の両方の健康と dysferlinopathy 患者線維芽細胞細胞トランスフェクション フルレングス ジスフェリン プラスミドの有無を評価するためのプロトコルが負傷した単純な二光子励起レーザーを概説します。

健康な線維芽細胞、dysferlinopathy の非投与患者線維芽細胞、フルレングス ジスフェリン シーケンス機能を再封止膜を評価するために負傷した二光子レーザーを受けたを含むプラスミドをトランスフェクトした患者線維芽細胞リアルタイムで.正常ひと線維芽細胞がレーザーが負傷した FM 4-64 蛍光活性の低レベルを表示し、非投与患者線維芽細胞展示相対蛍光強度損傷の...

Watch this Scientific Journal Video about Cell Membrane Repair Assay Using a Two-photon Laser Microscope at JoVE.com

Cell Membrane Repair Assay Using a Two-photon Laser Microscope

2 光子レーザー顕微鏡を用いた細胞膜修復法

2 光子レーザーを介して細胞膜が負傷した膜を再シール能力評価に広く使われている方法で、複数の細胞のタイプに適用できます。ここでは、体外でライブ イメージング dysferlinopathy 2 光子レーザー アブレーション患者細胞の膜を再シールするためのプロトコルについて述べる.

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

cell-membrane-repair-assay-using-a-two-photon-laser-microscope

Research

JoVE Journal

Immunology and Infection

12.3K ビュー.  University of Alberta Faculty of Medicine and Dentistry. 2 光子レーザーを介して細胞膜が負傷した膜を再シール能力評価に広く使われている方法で、複数の細胞のタイプに適用できます。ここでは、体外でライブ イメージング dysferlinopathy 2 光子レーザー アブレーション患者細胞の膜を再シールするためのプロトコルについて述べる.

ビデオ: Cell Membrane Repair Assay Using a Two-photon Laser Microscope

A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor

BACKGROUNDS: Previously, we demonstrated that neutralizing capacity but not the concentration of GM-CSF autoantibody was correlated with the disease severity in patients with autoimmune pulmonary alveolar proteinosis (PAP)1-3. As abrogation of GM-CSF bioactivity in the lung is the likely cause for autoimmune PAP4,5, it is promising to measure the neutralizing capacity of GM-CSF autoantibodies for evaluating the disease severity in each patient with PAP.
Until now, neutralizing capacity of GM-CSF autoantibodies has been assessed by evaluating the growth inhibition of human bone marrow cells or TF-1 cells stimulated with GM-CSF6-8. In the bioassay system, however, it is often problematic to obtain reliable data as well as to compare the data from different laboratories, due to the technical difficulties in maintaining the cells in a constant condition.
OBJECTIVE: To mimic GM-CSF binding to GM-CSF receptor on the cell surface using cell-free receptor-binding-assay.
METHODS: Transgenic silkworm technology was applied for obtaining a large amount for recombinant soluble GM-CSF receptor alpha (sGMR&alpha;) with high purity9-13. The recombinant sGMR&alpha; was contained in the hydrophilic sericin layers of silk threads without being fused to the silk proteins, and thus, we can easily extract from the cocoons in good purity with neutral aqueous solutions14,15. Fortunately, the oligosaccharide structures, which are critical for binding with GM-CSF, are more similar to the structures of human sGMR&alpha; than those produced by other insects or yeasts.
RESULTS: The cell-free assay system using sGMR&alpha; yielded the data with high plasticity and reliability. GM-CSF binding to sGMR&alpha; was dose-dependently inhibited by polyclonal GM-CSF autoantibody in a similar manner to the bioassay using TF-1 cells, indicating that our new cell-free assay system using sGMR&alpha; is more useful for the measurement of neutralizing activity of GM-CSF autoantibodies than the bioassay system using TF-1 cell or human bone marrow cells.
CONCLUSIONS: We established a cell-free assay quantifying the neutralizing capacity of GM-CSF autoantibody.

We designed a cell-free receptor binding assay in order to estimate the binding of granulocyte-macrophage colony-stimulating factor (GM-CSF) to the receptors. It enables us to evaluate competitive inhibition of biotinylated GM-CSF binding to soluble GM-CSF receptor alpha by GM-CSF autoantibody with excellent reproducibility.

組換え可溶性GM - CSF受容体を用いてGM - CSF抗体の中和能を推定する無細胞アッセイシステム

BACKGROUNDS: Previously, we demonstrated that neutralizing capacity but not the concentration of GM-CSF autoantibody was correlated with the disease ...

免疫・感染学

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

移植片対宿主病の誘導および<emインビボで&gt;</emMHCマッチしたマウスモデルを使用する&gt; T細胞のモニタリング

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

T6SS媒介細菌競争を監視するには、Visualアッセイ

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

メンブレン極性膜完全性、細胞内イオン濃度の監視の変更<em&gt;肺炎球菌</em&gt;蛍光染料を使用

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins are able to fuel the fusion process through their conformational changes could lead to the design of better, more effective immunogens for vaccine strategies. Here we describe a cell-based ELISA assay that allows studying the recognition of trimeric HIV-1 Env by monoclonal antibodies. Following expression of HIV-1 trimeric Env at the surface of transfected cells, conformation specific anti-Env antibodies are incubated with the cells. A horseradish peroxidase-conjugated secondary antibody and a simple chemiluminescence reaction are then used to detect bound antibodies. This system is highly flexible and can detect Env conformational changes induced by soluble CD4 or cellular proteins. It requires minimal amount of material and no highly-specialized equipment or know-how. Thus, this technique can be established for medium to high throughput screening of antigens and antibodies, such as newly-isolated antibodies.

Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.

細胞ベースのELISAアッセイを使用して、HIV-1エンベロープ糖タンパク質三量体の構造評価

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly synthesized allergic, inflammatory and immunoregulatory mediators. MCs&#8217; mediators affect multiple tissues and organs culminating in allergic and immune responses. The synthesis, storage and release of the MC mediators are highly regulated. The pre-formed mediators are packed in cytoplasmic secretory granules&#160;(SG) that fuse with the plasma membrane and release their content by regulated exocytosis. We present a protocol, based on the co-expression of a gene of interest with a reporter gene that is targeted to the SGs and is released in a regulated fashion alongside the endogenous SG mediators. The protocol enables high resolution four dimensional confocal analyses of the MC SGs and monitoring their timeline from biogenesis to triggered exocytosis. Thus, using this protocol for screening genes of interest for their phenotypic and functional impact allows deciphering the molecular mechanisms that govern the biogenesis and exocytosis of the MC SGs and identifying the regulators involved. Thereby, further insights into the cellular mechanisms that account for MCs function in health and disease should be provided.

The goal of the present&#160;protocol was to develop a method that will allow functional genomic analyses of mast cell secretion. The protocol is based on quantitative assessment of the release of a fluorescent reporter gene cotrasfected with the gene of interest and real time&#160;analyses of the secretory granule&#39;s morphology.

マスト細胞の分泌顆粒の調査。生からのエキソサイトーシスへ

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly ...

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, ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

網膜ミクログリアの貪食機能を評価します<em&gt;インビボ</em&gt;フローサイトメトリーベースのアッセイを使用しました

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

生体内で予防接種モデルを用いた抗原特異的 T 細胞細胞傷害性関数の検出のための試金

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

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

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 extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

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 ...