This work was supported by the Singapore National Research Foundation (grant NRF-RF2010-06 to J.Y.Y.).

1.試料の調製<ol><li> UAS-GCaMP5G 19ハエ（; P {20XUAS-IVS-GCaMP5G} attP40 1118ワット ）にGal4の 3,22ドライバーラインを通過。クロスは、25℃で成長させます。 注：生成は、蛍光シグナル強度を高めるために助けることができるのGal4またはUAS-GCaMP導入遺伝子の複数のコピーに飛びます。 Gr68a-Gal4ドライバーの制御下で発現された場合UAS-GCaMP導入遺伝子（UAS-GCaMP3）の以前のバージョンは、非常に微弱な蛍光強度を示しました。 <ol><li>収集し、分離セックス新たeclosed大人のハエによって、25℃で引き上げます。 注：GCaMP5Gの蛍光のベースラインレベルは14から28日の間熟成させハエにおける最適です。性別によってハエを分離することにより、神経応答に影響を与える可能性があり、社会的相互作用の可能性を排除します。 </li></ol></li><li> CO 2の穏やかな流れの下でハエを麻酔。 </li><li> Fを添付最初の0.17ミリメートルのカバースリップの中央にマニキュアの小滴を配置することにより、カバーガラスにLY。静かに明確なマニキュアの降下にその側にフライを配置し、全体の低下はハエで覆われていることを確認してください。 </li><li> 8X倍率で使用される解剖実体顕微鏡、下の手順1.5と1.6を実行します。 </li><li>ハエの前肢を拡張するために湿った絵筆を使用してください。第1,5足根のセグメント（ 図2A）の上にテープの2つの薄いストリップを配置することによって所定の位置に前足を固定します。これは、イメージング（ 図2B）のための足根セグメントT2-T4を公開します。 </li><li>口先上の画像ニューロンに、唇弁（ 図2C、D）を露出する吻の演壇の上にテープの薄いストリップを配置します。 </li><li>カバーガラスの表面上を流れるの解決策を防ぐために、PAPペンでフライの周りに疎水性の障壁を描画します。取り付けの30分以内に測定を行います。 </li></ol>刺激の調製溶液<ol><li>親油性リガンドを希釈PBST溶液中（例えば、本研究で使用CH503、など）：137のNaCl、2.7ミリモルのKCl、10mMののNa 2 HPO 4、1.8mMのKH 2 PO 4、0.1％トリトンX-100（v / v ）; pHは7.4。 <ol><li> PBSTを用いて、リガンドの1mg / mlの原液を作成します。 PBSTを用いて、原液のすべての後続の希釈液を調製します。 注：目的のリガンドの溶解性に応じて、異なる溶媒リガンドを溶解するために必要とされ得ます。種々の溶媒中でCH503の溶解度は、 表1に記載されている。他の溶媒と異なり、PBSTで蛍光の増加を生じませんでした。 </li></ol></li></ol>味覚ニューロンおよび画像取得の3刺激<ol><li> 10μlのピペッターを使用して、瞼のセグメント上にPBST10μlのを配置します。 注：このステップは、足を湿らし、漂流から足根セグメントを防ぎます。 </li><li> suコマンドのカメラを持つ光学系を使用してください高時間分解能に優れた蛍光シグナルを達成するためにfficient速度と感度。 注：この研究のために我々は（ 材料リストを参照してください）スピニングディスク共焦点顕微鏡とsCMOSカメラを使用していました。 </li><li>撮像されるべき特定の瞼板セグメントとニューロンを選択してください。 3刺激前の共焦点Z-スタックを取得します。 <ol><li>理想的には、まだ全体の細胞容積をキャプチャしながら、最大の時間分解能を可能にするのに十分に高速で取得設定を使用します。例取得設定は次のとおりです。200ミリ秒露出、2秒ごとに撮影し、×2 2と6×0.5μmの2光学切片をビニング。 注：固定化された脚はGCaMP信号の識別可能な漂白で、30秒間隔で、最大10分間正常に撮像されています。長時間録画のために、脚はカバースリップ上の所定の位置に非常に強固に保持されていることを確認してください。 </li><li>最高の信号対雑音全体IMA中に最小限の光退色で達成するためのレーザパワーを最適化します政権を銀杏。ここで説明する実験のために、脚と口吻にGr68aとppk23味覚ニューロンの両方を撮像するための30％の送信時に491 nmのダイオードレーザー（100ミリワット）を使用します。まだ十分な空間分解能を達成しながら、より短い露光時間を可能にするために2×2ビニングを使用してください。 注：レーザパワー設定値が1.2〜4.5の倍の増加の範囲の蛍光変化の検出を可能にするように最適化しました。 </li></ol></li><li>ピペット瞼のセグメントへの刺激溶液10μl。対照実験のために、PBSTの別の10μlを添加します。すぐに117刺激後の画像を取得します。 注：取得された刺激後画像の数は、蛍光の最大変化が達成された時間（約2分）に基づいています。 <ol><li>重要なステップ：これは前脚が所定の位置から移動しますので、準備にソリューションをピペット一方で、ピペットチップでフライに触れることはありません。 注：別の方法として、使用正確に結像される瞼のセグメントに近い刺激を含むガラスキャピラリーを配置するマイクロマニピュレーター。製造元の指示に従って、細胞内マイクロインジェクションディスペンサーを使用して、刺激の制御されたボリュームを提供します。 </li></ol></li></ol> 4.画像処理と解析：応答の定量<ol><li>フィジー（用いた共焦点Z-スタックの一連開き<a href="http://fiji.sc/Fiji" target="_blank">http://fiji.sc/Fiji</a> ）23またはImageJの（ <a href="http://imagej.nih.gov/ij/" target="_blank">http://imagej.nih.gov/ij/</a> ）24画像解析および処理ソフトウェアを。 </li><li> 120の時点のそれぞれについて、すべての6つのZスライスの和強度投影を行います。 <ol><li> 「イメージ」の下の「スタック」オプションを選択します。さらに、「Z投影」オプションを選択し、開始スライスとストップスライスとして「4」と「1」を入力します。投射型の場合は、Rを選択20;和スライスすべての時間枠」をプルダウンメニューから、とは選択します &quot;&quot;。 </li></ol></li><li>細胞体または神経突起の周りに境界線を描くようにフリーハンド選択ツールを使用して、関心領域（ROI）を定義します。 「分析」ラベルをクリックし、「ツール」の「ROIマネージャー」オプションを選択します。 </li><li> 「分析」メニューとの統合密度、面積、平均値、最大グレー値とラベルのボックスをチェックの下、「測定値の設定」オプションを選択することにより、ROIの蛍光を定量化します。そして、ROIマネージャのメニューの下の「詳細」オプションを選択し、さらに「マルチ測定」オプションを選択します。 注：これは、選択した各パラメータの値を持つポップアップボックスを生成します。 </li><li>ピクセルのいずれも（16ビットカメラ用に例えば 、65535）飽和状態に達していないことを確認するために、最大グレー値を確認してください。 注：トンに示す実験について彼の論文は、応答はΔF/ Fの最大増加が観察された場所に応じて、細胞体または神経細胞の突起のいずれかから測定しました。 </li><li>時刻tでの蛍光の最大相対変化（ΔF/ F）を計算し 、次のとおりです。 <img alt="式（1）" src="/files/ftp_upload/53392/53392eq1.jpg" /><ol><li>各F（セル）の値からバックグラウンド蛍光を引きます。バックグラウンド蛍光を定量化するために、検出可能なニューロンを欠い前足（または吻）の領域から同等のサイズと形状のROIの統合密度を測定。 </li><li>刺激後の蛍光を定量化するために、F（セル）T、時点における細胞体またはプロセスのROIの集積密度を測定し、「T」刺激後。 </li><li>予備刺激蛍光、F（細胞）、T = 0を定量化するために、同様に細胞体または工程の前刺激画像を解析ポスト刺激信号。 F（セル） トン = 0を決定するために3つの画像の平均統合密度を使用してください。 </li></ol></li></ol>

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The authors declare that there are no competing financial interests.

ここでは2つの異なる感覚器官にショウジョウバエ末梢ニューロンのライブカルシウムイメージングを実行する方法について説明します。 Ca 2+が CH503は、用量依存的かつ定量的であったフェロモンリガンドによって誘導されるGr68a-ニューロンにおけるGCaMP蛍光応答を-evoked。このような相性や強壮剤応答など、さまざまな神経応答パターンを識別することも可能でした。 <p class...

動物は生存と繁殖のための本質的な意思決定を媒介するために、嗅覚や味覚情報に依存しています。検出され、神経系によってどのように処理されるか化学感覚手がかり理解することは、感覚受容体（複数可）および対応する化学リガンドの同定を必要とする。 ショウジョウバエは、揮発性および非揮発性化合物の驚異的な多様性を検出し、生理を研究する中で優れたモデルですchemosensationのメカニズム。嗅覚器官が揮発性分子を認識しながら、味覚器官は低揮発性化合物を検出するために特化されます。ここでは、直接、低揮発性、親油性リガンドにキイロショウジョウバエの味覚器官からの神経細胞応答を測定するための方法を提示します。 ハエの味覚器官は前足、口吻、および翼を含みます。 sに応答する感覚器として知られている毛様構造は、味覚器官の表面に分布していますugars、ビター、塩、水およびフェロモン8。感覚子は味の剛毛と味ペグ9に形態学的に分類されています。ロング（L型）に分類されている唇弁上の約31味覚毛、ショート（Sタイプ）と中間（i型）形態があります。糖に生理的に応答する&#39;L&#39;と&#39;s&#39;の感覚器家4感覚ニューロン（S細胞）、低塩（L1細胞）、高塩と苦味化合物（L2細胞）と水（Wセル）10 、11。高塩12に他の応答しながら&#39;i&#39;を、低塩と砂糖の両方に応答するそのうちの一つ感覚子家2感覚ニューロンを、。男性の約41味覚感覚器と前足の各々の上に分布し、女性で26感覚器があります。男性と女性の両方のために、脚の中央部に21感覚器、および後肢13で約22感覚器があります。足の味覚感覚器で囲まれた味覚ニューロンはまた、cはL1、L2、WとSタイプにlassified。 単一ニューロンからの電気的活動を測定するための1つの標準的な方法は、イオンの流れを記録するために、細胞外または細胞内の電極を使用します。電極の測定は、神経機能は、 ショウジョウバエの種、蛾、および神経標識のために大規模な遺伝子ツールを欠いているミツバチなどの非モデル生物で研究することを可能にします。しかし、電気生理学的方法は、日常的に、このアプローチを適用し、 ショウジョウバエの味覚感覚器4,13,14の活性を測定するために使用されてきたが、いくつかの技術的な課題を提示します。まず、味の剛毛は形態および空間的な位置に基づいて特定する必要があります。識別されると、電気生理学的測定は、感覚器の小型化、による位置に制限のアクセシビリティ、および毛の味に化学的刺激の制御されたボリュームを適用することの難しさによって妨げことができます。また、感覚器を刺激は、複数の信号を生成することができますニューロンのタイプ15。第二、電気信号​​の検出は、機械的振動や電子機器からのノイズに起因するバックグラウンドノイズによって混乱することができます。誤っ14を使用する場合第三に、先鋭化電極の使用は、フライの準備を損傷する可能性があります。最後に、電気生理学リグを組み立てると、刺激送達、信号記録、及びデータ分析のための特殊な電子部品を必要とし、高価であることができます。 D.でショウジョウバエ 、遺伝的にコードされたツールの利用可能性は、ニューロンの小集団の応答を検討することを可能にする画像化技術の開発が容易になりました。このようなアプローチの1つは、CaLexAレポーター16を使用することです。この方法では、のLexA-VP16転写因子及びカルシウム感受性タンパク質NFATをコードする遺伝子配列が一緒に融合されます。プロテインホスファターゼカルシニューリンのカルシウム活性化は、NFATの脱リン酸化を触媒し、順番に、facilita核へのインポートをTES。核内部に、LexAのドメインは、このように機能的に活性化されたニューロンの持続的な同定を可能にする、緑色蛍光タンパク質（GFP）レポーターの発現を導くLexAop-DNA結合モチーフに特異的に結合します。アプローチは、付臭剤16へのライブハエの暴露後の触角葉における特定の嗅覚糸球体の応答を測定するために成功裏に使用されてきました。最近、IR52c味覚受容体ニューロンの生理学的反応はCaLexA 7を使ってライブハエから測定しました。しかしながら、この研究のために、それは、GFPの転写を増強するために最大6週間齢のハエに必要でした。抗GFP抗体で免疫染色するCaLexA信号をブーストするために使用することができるが、この方法は、このように生細胞イメージングの可能性を排除し、組織の固定を必要とするであろう。 遺伝的にコードされたカルシウム指標タンパク質GCaMPも広範囲の数の神経細胞応答を研究するために使用されてきました種17。タンパク質GCaMPは、神経刺激の前に、低強度で蛍光を発します。刺激の適用は、Ca 2+の流入で、その結果、神経細胞に活動電位をトリガします。 Ca 2+に結合したGCaMPは、それが明るい強度（ 図1）と蛍光を引き起こし、コンフォメーション変化を受けます。最近開発された単一ニューロンカルシウムイメージング法は、前足特定Gr61aの味覚ニューロン18のリガンドとしてグルコースを同定しました。本研究では、Gr61aの味覚ニューロンにおいてGCaMPを発現するトランスジェニックショウジョウバエから解剖前足は、撮像前にアガロースの層で覆われていました。しかしながら、解剖組織の使用は、測定時間と検出感度を制限し、GCaMP発現の最終的荒廃を引き起こすことができます。また、アガロースは、高い蛍光のバックグラウンドレベルとその光散乱特性に対する感度を制限することができます。 TOこれらの欠点のいくつかを解決、我々は前脚と口吻無傷の動物の味覚ニューロンからの生理的反応を記録するGCaMP媒介カルシウムイメージングの使用を記載しています。我々は（3 R、11 Z、19 Z）-3-アセトキシ-11,19-octacosadien -1-オール（CH503）20、 ショウジョウバエの脂質フェロモンに遺伝的にコードされGCaMP5G 19を表現Gr68aとppk23ニューロンの生理学的応答を示します21。ニューロンの応答は、フェロモン刺激時GCaMP5G信号の蛍光の増加を定量することによって測定されます。このプロトコルでは、ニューロンは、個々の細胞内の神経活動のパターンを区別するのに十分である120秒の総持続時間のために結像されます。

<table>
	<tbody>
		<tr>
			<td>Gr68a-Gal4</td>
			<td></td>
			<td></td>
			<td>Gift from H. Amrein (Texas A&#38;M Health Science Center, TX, USA) and J. Carlson (Yale University, CT, USA)</td>
		</tr>
		<tr>
			<td>ppk23-Gal4</td>
			<td></td>
			<td></td>
			<td>Gift from K. Scott (Univ. of California, Berkeley, CA, USA)</td>
		</tr>
		<tr>
			<td>UAS-GCaMP5&#160;</td>
			<td></td>
			<td>42037</td>
			<td>Bloomington Drosophila&#160; Stock Center</td>
		</tr>
		<tr>
			<td>0.17 mm coverslip (Gold-Seal coverslip)</td>
			<td>Electron Microscopy Services</td>
			<td>63790-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Nail polish, &#34;Hard as Nails Clear&#34;&#160;</td>
			<td>Sally Hansen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PAP pen</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>Z377821</td>
			<td></td>
		</tr>
		<tr>
			<td>Paint brush</td>
			<td></td>
			<td></td>
			<td>fine-tipped brush</td>
		</tr>
		<tr>
			<td>Tape&#160;</td>
			<td>Scotch brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>13021</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, lab grade</td>
			<td>Merck</td>
			<td>10094</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexane, HPLC grade</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>H303SK-4</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>472301</td>
			<td></td>
		</tr>
		<tr>
			<td>PBST</td>
			<td></td>
			<td></td>
			<td>Recipe described in the protocol section</td>
		</tr>
		<tr>
			<td>CH503</td>
			<td></td>
			<td></td>
			<td>Synthesis described in Mori et al., 2010</td>
		</tr>
		<tr>
			<td>sCMOS Camera (ORCA Flash4.0)</td>
			<td>Hamamatsu&#160;</td>
			<td>C11578-22U</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope (Ti-Eclipse)</td>
			<td>Nikon</td>
			<td>Ni-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinning Disk Scan head&#160;</td>
			<td>Yokogawa</td>
			<td>CSU-X1-A1</td>
			<td></td>
		</tr>
		<tr>
			<td>Aquistion Software (MetaMorph Premier)</td>
			<td>Molecular Devices</td>
			<td>40002</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji software</td>
			<td>open source</td>
			<td></td>
			<td><a href="http://fiji.sc/Fiji">http://fiji.sc/Fiji</a></td>
		</tr>
	</tbody>
</table>

measuring physiological responses of drosophila sensory neurons to lipid pheromones using live calcium imaging

哺乳類とは異なり、このようなショウジョウバエなどの昆虫は、複数の味覚器官を持っています。脚、吻、翼とショウジョウバエの産卵管上の化学感覚ニューロンは、味覚受容体1,2、イオンチャンネル3-6、揮発性および不揮発性の感覚手がかりの検出に関与するイオンチャネル型受容体7を発現します 。これらのニューロンは、直接、食品、有害物質やフェロモンのような味物質に接触し、したがって、このような給紙、産卵や交配などの多くの複雑な動作に影響を与えます。電極記録とカルシウムイメージングは​​、広くこれらの味物質によって誘発される神経細胞の応答を定量するために、昆虫に使用されてきました。しかし、電気は特殊な装置を必要とし、単一の味感覚子からの測定値を得ることは、細胞型、サイズ、および位置に応じて技術的に困難であることができます。また、 ショウジョウバエにおける単一ニューロン分解能は味の感覚器の侯ので、達成することが困難な場合が化学感覚ニューロンの複数のタイプがSE。ここに記載のライブカルシウムイメージング法は、ライブハエにおける単一味覚ニューロンの応答を測定することができます。この方法は、水性溶媒中で低い溶解度を有する脂質フェロモンおよび他のリガンドタイプのニューロンの応答を画像化するために特に適しています。

GCaMPのカルシウム指示薬を遺伝子Gr68a-GAL4またはppk23-Gal4のドライバを使用して発現させました。前脚の神経細胞および非神経支持細胞の異なる集団は、各ドライバ（ 図3A-C）によって標識されています。親油性フェロモンCH503へのリガンド特異的応答はGr68a-GAL4とGCaMP（ 図3D）を発現ppk23-Gal4の細胞で観察されました。 GCaMP5G信号（ΔF/ F）...

Watch this Scientific Journal Video about Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging at JoVE.com

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

の生理反応を測定します<em&gt;ショウジョウバエ</em&gt;ライブカルシウムイメージングを使用して脂質フェロモンの感覚ニューロン

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

measuring-physiological-responses-drosophila-sensory-neurons-to-lipid

Research

JoVE Journal

Immunology and Infection

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

8.5K ビュー.  Temasek Life Sciences Laboratory. The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone. 

ビデオ: Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

全葉のイメージングを使用して細菌細胞をライブに気孔反応を評価する

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

免疫・感染学

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

に感染したマウスにおける細菌負荷と免疫応答の測定リステリア菌</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

の生細胞イメージング枯草菌と肺炎球菌自動タイムラプス顕微鏡を使用して

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

ライブセル蛍光顕微鏡を用いた細菌毒素誘導される応答の可視化

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

Advances in live cell fluorescence microscopy techniques, as well as the construction of recombinant viral strains that express fluorescent fusion proteins have enabled real-time visualization of transport and spread of alphaherpes virus infection of neurons. The utility of novel fluorescent fusion proteins to viral membrane, tegument, and capsids, in conjunction with live cell imaging, identified viral particle assemblies undergoing transport within axons. Similar tools have been successfully employed for analyses of cell-cell spread of viral particles to quantify the number and diversity of virions transmitted between cells. Importantly, the techniques of live cell imaging of anterograde transport and spread produce a wealth of information including particle transport velocities, distributions of particles, and temporal analyses of protein localization. Alongside classical viral genetic techniques, these methodologies have provided critical insights into important mechanistic questions. In this article we describe in detail the imaging methods that were developed to answer basic questions of alphaherpes virus transport and spread.

Live cell imaging of alphaherpes virus infections enables analysis of the dynamic events of directed transport and intercellular spread. Here, we present methodologies that utilize recombinant viral strains expressing fluorescent fusion proteins to facilitate visualization of viral assemblies during infection of primary neurons.

Alphaherpesウイルス順行性輸送とスプレッドの生細胞イメージング

Advances in live cell fluorescence microscopy  techniques, as well as the construction of recombinant viral strains that  express fluorescent fusion ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

ファージフェノミクス：小説のウイルスタンパク質を特徴づけるために、生理的アプローチ

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

アン<em&gt;インビトロ</em&gt; HIV重感染のコンテキストでのマラリアに対する免疫応答を測定するためのモデル

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of lipids. The function of lipids (e.g., in host-pathogen interactions or host entry) has been reported to play a crucial role in cellular processes. Here, we show a method to determine lipid composition, with a focus on the cholesterol level of primary blood-derived neutrophils, by HPTLC in comparison to high performance liquid chromatography (HPLC). The aim was to investigate the role of lipid/cholesterol alterations in the formation of neutrophil extracellular traps (NETs). NET release is known as a host defense mechanism to prevent pathogens from spreading within the host. Therefore, blood-derived human neutrophils were treated with methyl-&#946;-cyclodextrin (M&#946;CD) to induce lipid alterations in the cells. Using HPTLC and HPLC, we have shown that M&#946;CD treatment of the cells leads to lipid alterations associated with a significant reduction in the cholesterol content of the cell. At the same time, M&#946;CD treatment of the neutrophils led to the formation of NETs, as shown by immunofluorescence microscopy. In summary, here we present a detailed method to study lipid alterations in neutrophils and the formation of NETs.

Lipids are known to play an important role in cellular functions. Here, we describe a method to determine the lipid composition of neutrophils, with emphasis on the cholesterol level, by using both HPTLC and HPLC to gain a better understanding of the underlying mechanisms of neutrophil extracellular trap formation.

脂質好中球の変化と好中球細胞外トラップのその後の形成を研究する方法

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

試金を使用して SIAT1 MDCK 細胞のインフルエンザ a (h3n2) ウイルス中和によるひと血清中の抗体を中和することを測定

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ cytosolic concentrations, linked to release from internal stores or an influx through plasma membrane channels, regulate cortical cell processes. Pathogens that adhere to or invade host cells trigger a reorganization of the actin cytoskeleton underlying the host plasma membrane, which likely affects both global and local Ca2+ signaling. Because these events may occur at low frequencies in a pseudo-stochastic manner over extended kinetics, the analysis of Ca2+ signals induced by pathogens raises major technical challenges that need to be addressed.
Here, we report protocols for the detection of global and local Ca2+ signals upon a Shigella infection of epithelial cells. In these protocols, artefacts linked to a prolonged exposure and photodamage associated with the excitation of Ca2+ fluorescent probes are troubleshot by stringently controlling the acquisition parameters over defined time periods during a Shigella invasion. Procedures are implemented to rigorously analyze the amplitude and frequency of global cytosolic Ca2+ signals during extended infection kinetics using the chemical probe Fluo-4.

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Here, we present protocols to visualize calcium (Ca2+) responses elicited by HeLa cells infected by Shigella. By optimizing the parameters of bacterial infection and imaging with Ca2+ fluorescent probes, atypical global and local Ca2+ signals induced by bacteria over a large range of infection kinetics are characterized.