The authors thank Ian Clarke and P. Scott Hefty for the pGFP-SW2 and pASK-GFP-mKate2-L2 plasmids, respectively. We thank Paul Miller for technical assistance and Richard Stephens for other resources. This work was funded by NIH grant AI095603 (KH).

蛍光ホスト細胞株の1世代<ol><li> 2×10 の 6細胞/ウェル6ウェルプレートに1日目プレート293T細胞（または他のレトロウイルスパッケージングライン）、〜75％コンフルエント翌日にします。必要に応じて、各レトロウイルス用プレートの重複ウェルを使用します。 </li><li> 2日目を吸引細胞と2 mlを加え、新鮮な増殖培地（DMEM + 10％FBS + 2mM L-グルタミン）。リポフェクタミン2000または同様の試薬 ​​を用いて、製造業者の指示に従って5-8μgのレトロウイルスベクター（GFPを含む）、パッケージングベクターのそれぞれ（ 例えば 、pVSVg）で細胞をトランスフェクトします。 </li><li> 37℃のCO 2インキュベーターで4〜6時間のためのDNAで細胞をインキュベートし、続いて1ミリリットルの成長培地で培地を交換してください。 </li><li> 37℃のCO 2インキュベーター中で 48時間、細胞をインキュベートします。 </li><li>ウェル10 5細胞 /のおおよその密度で6ウェルプレート上で3日目プレートの標的細胞（ 例えば 、HeLa細胞）、〜50％コンフルエントであるために次の日。倒立顕微鏡でGFP蛍光をモニターすることにより、293T細胞で陽性トランスフェクションを確認してください。 </li><li> 4日目は、ピペットを用いて293T細胞からレトロウイルス含有上清を収集し、0.45μmの酢酸セルロースシリンジフィルターを通して上清をフィルタリングします。 </li><li> HeLa細胞から培地を除去し、16 / mlのポリブレンを含む0.5 mlの増殖培地を追加します。賢明ドロップ、24時間、37℃のCO 2インキュベーターで細胞を培養する細胞にレトロウイルスのフィルタリング〜0.5ミリリットルを追加します。 4℃で（重複ウェルからすなわち 、）残りのレトロウイルスを保管してください。 </li><li>二連のウェルは、余分なレトロウイルスを生成するために行われた場合は5日目には、シリアル残りのレトロウイルス粒子を感染させるためにステップ1.7を繰り返します。 </li><li>選択抗生物質（例えば、500 / mlのネオマイシン）を含む10cmの皿に1：6日目通過は、HeLa細胞1をトランスフェクトしました。 </li><li>形質導入した細胞は、選択antibioの存在に戻って成長するのに十分な時間を待って細胞は、選択抗生物質（ 例えば 、200 / mlのネオマイシン）の低い濃度で、標準的な技術によって増殖させることができ、その後、チック。 注：必要に応じて、細胞はまた、クローン、この時点で理想的な明るさのために選択することができます。 </li></ol> GFP発現クラミジアトラコマティスの2世代<ol><li> 2倍のCaCl 2バッファー（20 mMトリスpHは7.4、100 mMのCaCl 2を）と4SPバッファー（0.4 Mショ糖、16mMのの Na 2 HPO 4）を準備します。 </li><li> 1日目解凍冷凍C.トラコーマ株式氷上で、すぐに50μlの2倍のCaCl 2バッファーに10μlの細菌を希釈。よく混ぜ、3μgのプラスミドDNA（ 例えば 、PASK-GFP-mKate2-L2）を追加し、25℃で30分間インキュベートします。 </li><li>このステップの間に、遠心管にMcCoy細胞のT-75フラスコをトリプシン処理することにより、宿主細胞を準備します。 5分間50×gで細胞懸濁液を遠心し、上清を捨てます。 Resuspen1XのCaCl 2緩衝液の適切な容積中のd細胞ペレットを4×10 7細胞/ mlの最終濃度を得ました。 </li><li> （ステップ2.2）から30分間インキュベートした後、形質転換混合物管（総体積200μlの）にMcCoy細胞を用意100μlを添加し、25℃でさらに20分間インキュベートします。 2ミリリットルの成長培地で6ウェルプレートとオーバーレイから単一のウェルに、この形質転換細胞の混合物の100μlのを追加します。 2日間、CO 2インキュベーター中、37℃でインキュベートします。 </li><li> 3日目を溶解McCoy細胞は 、Cに感染しました1ミリリットルのdH 2 Oで培地を交換し、曲がっ千μlのピペットチップを用いて細胞を掻き取ってトラコーマ 。 Cの効率的な細胞溶解および放出のための5回1ミリリットル4SPバッファを追加して、〜27.5 G針を通してサスペンションを渡しますトラコーマ 。 </li><li> C.を含む細胞溶解物を2mlの転送たてメッキMCCのコンフルエントな単層を含むT-75フラスコにトラコマチスオイ細胞; （FBSなし）8 mlのDMEMを添加し、Cを可能にするために25℃で2時間インキュベート細胞に付着するトラコーマ 。 </li><li>吸引し細胞および10 U / mlのペニシリンGと1 / mlのシクロヘキシミドを補充した新鮮な増殖培地を追加します。 37℃のCO 2インキュベーター中で 48時間、フラスコをインキュベートします。 </li><li> 4日目は、細胞が感染していると介在物が異常クラミジアが含まれている光学顕微鏡で確認してください。標準の光学顕微鏡に明るい空胞、および直径2μm以上である介在物内にリング状の物体などの異常クラミジア体として介在物を特定します。 </li><li> 2ミリリットルのdH 2 Oで培地を交換し、懸濁液中に細胞をこすり取ることにより、5日目を溶解文化。 2ミリリットル4SPバッファを追加し、約5倍の27.5 G針を通してサスペンションを渡します。 </li><li> 6ウェルプレート中で単一ウェルでMcCoy細胞の新鮮​​な単層を感染させるために、細胞溶解物の2ミリリットルを使用してください。 25℃で2時間溶解液で細胞をインキュベートし、吸引し、新鮮な成長を追加ペニシリンGおよびシクロヘキシミドを含む培地。 </li><li>細胞の一般的な健康状態に応じて、3〜5日間、37℃のCO 2インキュベーター中で細胞をインキュベートします。 </li><li>繰り返し（異常な体を欠いている）通常の探している介在物が出現するまで、6ウェルプレート中の新鮮なウェルに2.9から2.10 1回以上、必要に応じて、継代培養を繰り返します。この時点でことを確認するために倒立蛍光顕微鏡を使用するC.トラコーマは mKate2（赤）を発現します。 </li><li> T-150フラスコ中で増殖させ、感染したMcCoy細胞またはHeLa細胞からのクラミジアの形質転換体を採取することにより、たとえば、高力価のクラミジア株を生成するため、感染した培養物をスケールアップ。 </li></ol> 3. クラミジアで細胞を感染させます<ol><li> IFUの決意とマルチウェルスクリーニングのための、例えばガラスボトムディッシュまたはチャンバースライドの高解像度画像化のために、または24ウェルプレートのための所望の培養容器にプレートGFP-HeLa細胞、。 </li><li>あちこち解凍Cを含む禅チューブトラコマティス、C。 muridarum、またはC.氷上ニューモニエ例えばMOI = 1、感染の所望の多重度（MOI）にHBSS中に希釈します。 </li><li>使用される特定のクラミジア株に基づいて静的または遠心支援の感染症のいずれかを実行します。 <ol><li> C.での感染についてトラコマチス LGV血清型L2またはC. muridarumは 、HBSSでGFP-HeLa細胞を洗浄し、25℃で2時間希釈した細菌とインキュベートします。吸引し、HBSSで細胞を洗浄し、37℃のCO 2インキュベーター中で増殖培地中で細胞をインキュベートします。 </li><li> C.トラコマチス血清型DまたはCと感染症肺炎連鎖球菌は 、HBSSでGFP-HeLa細胞を洗浄し、細胞に希釈した細菌を追加します。卓上遠心機で、スイングバケットロータ取付のマルチウェルプレートホルダーにマルチウェルプレートまたは（マルチウェルプレートの蓋にテープで固定）ガラスボトムディッシュを置きます。 </li><li> 1時間25℃で900×gで遠心分離した細胞。 </lI&gt; <li> 1時間、37℃のCO 2インキュベーター中で遠心分離し、場所から培養容器を外します。 </li><li>安全キャビネット内を吸引細胞は、HBSSで細胞を2回洗浄し、新鮮な増殖培地を追加します。 37℃のCO 2インキュベーター内で場所細胞。 </li></ol></li></ol> クラミジア介在物の4生細胞の可視化<ol><li> CO 2インキュベーターからクラミジア感染GFP-HeLa細胞を削除し、フェノール5 +赤％のFBSを含まないRPMIで培地を交換してください。 </li><li> 20倍以上のオイル対物レンズを用いて倒立蛍光顕微鏡（ニコンエクリプスのTi-Eまたは類似の）上のマウント細胞。 </li><li>大きなブラックホール（ クラミジア介在物）を含む緑色の細胞：イメージングソフトウェア（Volocity 6または類似）を使用して、に焦点を当て、特定クラミジアは、GFP-HeLa細胞を感染させました。 </li><li>マークイメージングソフトウェアを使用して、関心のクラミジアに感染した細胞を含む領域のXYZ場所（巻ocity 6または類似）。手動XYステージ表示モードでしばらく「ポイントの追加」をすることによって、これを実行します。このように、XYZ座標は、各マークされた場所のために保存されています。 </li><li>取得設定]ダイアログボックスで、緑（GFP、ヒーラ細胞質ゾル）と赤（mKate2、 トラコーマクラミジア ）チャネル、およびチャネルごとに新しいフレームごとに5分の割合でを取得するソフトウェアを設定します。 </li><li>取得プロトコルを使用して画像シーケンスを取得」キャプチャ/録音」ボタンをクリックすることで、上記で入力しました。 </li></ol>生細胞内封入成長パラメータの5定量<ol><li>所望の時間感染後（ 例えば 、24、48 HPI）でインキュベーターからクラミジア感染GFP-HeLa細胞を除去し、倒立蛍光顕微鏡に取り付けます。注：高NAの20Xや40X空気目的は、24ウェルプレートのために最も適しており、40X油目的は、チャンバースライドをお勧めします。このお尻のためのいずれかの基板上に細胞を増殖させますAY。 </li><li>介在物は、その最大径であり、鮮明なエッジをもたらす細胞のミッドプレーン、に焦点を当てています。 </li><li>各ウェル（ウェル当たり少なくとも10）に多くの分野のための画像を取得します。ウェルは、通常、複製または実験変数を表します。 </li><li>目で（介在物は、GFP-HeLa細胞における黒区画されている）、手動で介在物の個数を列挙したり、自動的に介在物を特定し、カウントするイメージングソフトウェア・アルゴリズムを使用しています。 <ol><li>蛍光強度によって、GFP-HeLa細胞を探す（コマンド「オブジェクトの検索」）を、細胞の相対強度に基づいて、蛍光閾値強度を調整します。 </li><li> 52μm以下だけ大きいものを維持するサイズのガイドラインを使用して、選択したオブジェクトをフィルタリングします。これは、「人口1」です。 </li><li>入力として「人口1 &#39;を使用して、コマンド「オブジェクトの穴フィル」を適用します。このステップは、「人口2 &#39;を生成します。 </li><li> P &#39;から「人口1」を減算し「人口3 &#39;と指定積極マークさクラミジア介在物を、生成する「opulation 2。 </li><li>例えば、24時間以上のために感染した細胞内封入体のためには25μm2よりも大きなオブジェクトを保持するために、「人口3 &#39;（コマンド「人口フィルター」を）にサイズのフィルタを適用します。これらの介在物がはるかに小さいなどの前に18時間早ければ感染の時間については、設定サイズのフィルタは、4μm2でより大きなオブジェクトを保持します。 「介在物」として指定されたオブジェクトの集団でのサイズフィルタリング結果。 </li><li>イメージングソフトウェアを使用して、例えば、画像から封入番号、封入サイズ、及び包含外周の定量的データを計算します。 </li></ol></li></ol>

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著者は、彼らが競合する金融利害関係を有していないことを宣言します。

ここでは、リアルタイムの可視化とクラミジア介在物の分析のための蛍光宿主細胞を生成するための実験的な戦略について説明します。この液胞の可視化手法は、点灯トラックと定量的に細胞の集団間または経時単一細胞におけるクラミジア介在物の動的特性を測定するための強力な能力を付与する。蛍光タンパク質標識細胞におけるクラミジア介在物が著しく十分に定義され?...

クラミジアは性感染症、骨盤内炎症性疾患、失明、肺炎、おそらくアテローム性動脈硬化症1-4を含む、世界の健康に大きな負担を惹起細胞内細菌の種によって引き起こされる感染症。液胞の中から、宿主細胞と相互作用するクラミジアの能力は、（封入と呼ばれる）、それらの成功した細胞の感染および宿主のための重要な決定要因です。包含は、クラミジアの成長を可能にし、動的にクラミジア 5の全体の2〜3日の発達サイクルを通じて変更された新たな病原性の区画です。クラミジアの偏性細胞内性質は直接含めることのユニークな生物学を研究するため、特に研究コミュニティに多くの課題を提示。主要なハンディキャップを効率的に蛍光アプローチによって、細胞内のクラミジアやその含有物のいずれかを可視化することができないことでした生きた細胞内のES。最近の発見は、Cを GFP発現を生成するための手段を最終的に明らかにしましたトラコーマ 6;しかし、この発見は、まだ包含の特異的な標識には至っていません。いくつかの技術は、細菌や介在物7,8の標識に記載されているが、彼 ​​らは、このような光退色に対して非特異性、仮初や感受性などの欠点があります。私たちのグループによる重要な発見は、宿主細胞9 GFP発現を使用して含めることを照明するための新たな戦略を確立しました。この戦略は、合理的にダ10 520よりも大きい分子に包接膜の固有の不透過性を利用します。細胞を安定的に特定の細胞質の蛍光タンパク質（例えば、GFPまたはmCherryを）を発現するように操作された場合、 クラミジア介在物は、蛍光の彼らの完全な排除による著しい明快で見ることができます。この逆イメージング戦略は、すべてのクロロフィルのための介在物の即時の可視化を可能にしますamydia種と容易に関心のほとんど宿主細胞に適合させることができます。その有用性の実証として、この方法は、 クラミジア属 9のための細胞の出口経路を明らかにし、定義するために以前に使用されました。 ここでは、さらにこの方法が行われ、包接成長のダイナミクスについての重要な定量的データを得るために活用することができる方法を示しています。また、効果的にコストのかかる抗体ベースの列挙法の代わりにすることができ、そのようなクラミジア 11 mKate2発現などの他の蛍光標識と並行して使用することができます。ツールのこの強力な組み合わせは、宿主細胞を生きた内部クラミジア封入体膜の物性の探査を可能にします。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-Mem</td>
			<td>Invitrogen</td>
			<td>31985</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Sigma</td>
			<td>H9268</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 &#181;m filters</td>
			<td>Fisher</td>
			<td>09-719D</td>
			<td></td>
		</tr>
		<tr>
			<td>G418</td>
			<td>Invitrogen</td>
			<td>10131</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Invitrogen</td>
			<td>14025</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>11995</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Invitrogen</td>
			<td>11875</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 w/o phenol red</td>
			<td>Cellgro</td>
			<td>17-105-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin G</td>
			<td>Sigma</td>
			<td>13752</td>
			<td></td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma</td>
			<td>C7698</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottom dishes</td>
			<td>MatTek</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Chamber slides, Lab-Tek II</td>
			<td>Nunc</td>
			<td>154526, 154534</td>
			<td></td>
		</tr>
	</tbody>
</table>

using fluorescent proteins to visualize and quantitate chlamydia vacuole growth dynamics in living cells

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

サイトゾル蛍光タンパク質 （例えば、GFP）を発現する哺乳動物細胞を生、感染細胞培養物においてクラミジア介在物の照明を可能にするように設計することができます。 クラミジアの感染の際に、介在物は、宿主細胞（ 図1）における黒点として容易に見ることができます。蛍光欠けている介在物の透明度は、ビューおよび/ ​​または治療（ 図1）</st...

Watch this Scientific Journal Video about Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells at JoVE.com

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

視覚化し、定量化するために蛍光タンパク質を使用しました<em&gt;クラミジア</em生細胞内&gt;液胞の成長ダイナミクス

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

using-fluorescent-proteins-to-visualize-quantitate-chlamydia-vacuole

Research

JoVE Journal

Immunology and Infection

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

7.5K ビュー.  University of Washington. A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

ビデオ: Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

フローサイトメトリーで固定し、生細胞におけるカルパイン活性の測定

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

免疫・感染学

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

細胞星状に7つのステップ

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

細胞運命を以下に<em&gt; E大腸菌の</emラムダファージによる感染後&gt;

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium bacterial colonies growing inside tumors. 
We generated model xenograft tumors in mice by subcutaneous injection of a human ovarian cancer cell line, OVCAR-8 (NCI DCTD Tumor Repository, Frederick, MD). 
We transformed attenuated strains of S. typhimurium bacteria (ELH430:SL1344 phoPQ- 1) with a constitutively expressed luciferase (luxCDABE) plasmid for visualization2. These strains specifically colonize tumors while remaining essentially non-virulent to the mouse1. 
Once measurable tumors were established, bacteria were injected intravenously via the tail vein with varying dosage. Tumor-localized, bacterial gene expression was monitored in real time over the course of 60 hours using an in vivo imaging system (IVIS). At each time point, tumors were excised, homogenized, and plated to quantitate bacterial colonies for correlation with gene expression data. 
Together, this data yields a quantitative measure of the in vivo growth and gene expression dynamics of bacteria growing inside tumors.

Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium bacterial colonies growing inside tumors. This video covers tumor cell preparation and implantation, bacteria preparation and injection, whole-animal luminescence imaging, tumor excision, and bacterial colony counting.

腫瘍標的の測定成長と遺伝子発現ダイナミクス<em&gt; S.ネズミチフス菌</em&gt;バクテリア

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium ...

Forward Genetic Approaches in Chlamydia trachomatis

Chlamydia trachomatis, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its intractability to experimental transformation with recombinant DNA. We developed an approach to perform genetic analysis in C. trachomatis&#160;despite the lack of molecular genetic tools. Our method involves: i.) chemical mutagenesis to rapidly generate comprehensive libraries of genetically-defined mutants with distinct phenotypes; ii.) whole-genome sequencing (WGS) to map the underlying genetic lesions and to find associations between mutated gene(s) and a common phenotype; iii.) generation of recombinant strains through co-infection of mammalian cells with mutant and wild type bacteria. Accordingly, we were able to establish causal relationships between genotypes and phenotypes. The coupling of chemically-induced gene variation and WGS to establish correlative genotype&#8211;phenotype associations should be broadly applicable to the large list of medically and environmentally important microorganisms currently intractable to genetic analysis.

Forward Genetic Approaches in Chlamydia trachomatis

We describe a methodology to perform genetic analysis in Chlamydia based on chemical mutagenesis and whole genome sequencing. In addition, a system for DNA exchange within infected cells is described that can be used for genetic mapping. This method may be broadly applicable to microbial systems lacking transformation systems and molecular genetic tools.

フォワード遺伝的アプローチで<em&gt;クラミジアトラコマチス</em

Chlamydia trachomatis, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its ...

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 ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

アフリカトリパノソーマにグライコダイナミクスを監視する蛍光タンパク質を使用した

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force for plasma membrane extension around the particle. In addition, efficient engulfment of large material relies on focal exocytosis of intracellular compartments. This process is highly dynamic and numerous molecular players have been described to have a role during phagocytic cup formation. The precise regulation in time and space of all of these molecules, however, remains elusive. In addition, the last step of phagosome closure has been very difficult to observe because inhibition by RNA interference or dominant negative mutants often results in stalled phagocytic cup formation. 
We have set up a dedicated experimental approach using total internal reflection fluorescence microscopy (TIRFM) combined with epifluorescence to monitor step by step the extension of pseudopods and their tips in a phagosome growing around a particle loosely bound to a coverslip. This method allows us to observe, with high resolution the very tips of the pseudopods and their fusion during closure of the phagosome in living cells for two different fluorescently tagged proteins at the same time.

&quot;Phagosome Closure Assay&quot; to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM

We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

「ファゴソーム閉鎖アッセイ」全反射蛍光顕微鏡（TIRFM）を用いて、三次元的にファゴソーム形成を可視化します

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

精密カットライブ肺樹状細胞を可視化するために、マウスの肺スライスを

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

Studying Organelle Dynamics in B Cells During Immune Synapse Formation

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both signaling&#160;and antigen uptake are coordinated. IS formation involves dynamic actin remodeling accompanied by the polarized recruitment to the synaptic membrane of the centrosome and associated intracellular organelles such as lysosomes and the Golgi apparatus. Initial stages of actin remodeling allow B cells to increase their cell surface and maximize the quantity of antigen-BCR complexes gathered at the synapse. Under certain conditions, when B cells recognize antigens associated to rigid surfaces, this process is coupled to the local recruitment and secretion of lysosomes, which can facilitate antigen extraction. Uptaken antigens&#160;are internalized into specialized endo-lysosome compartments for processing into peptides, which are loaded onto major histocompatibility complex II (MHC-II) molecules for further presentation to T helper cells. Therefore, studying organelle dynamics associated with the formation of an IS is crucial to understanding how B cells are activated. In the present article we will discuss both imaging and a biochemical technique used to study changes in intracellular organelle positioning and cytoskeleton rearrangements that are associated with the formation of an IS in B cells.

Herein we describe two approaches to characterize cell polarization events in B lymphocytes during the formation of an IS. The first, involves quantification of organelle recruitment and cytoskeleton rearrangements at the synaptic membrane. The second is a biochemical approach, to characterize changes in composition of the centrosome, which undergoes polarization to the immune synapse.