The authors are financially supported by the Swiss National Science Foundation through an operating grant N&#176; 31003A-149566 (to N.D.), and The Sir Jules Thorn Charitable Overseas Trust through a Young Investigator Subsidy (to P.N.).

倫理の声明：すべての動物の操作は、ジュネーブ大学の動物実験委員会のガイドラインに厳密に従って行きました。 貪食ターゲットの作製<ol><li> 10 mlの滅菌リン酸緩衝食塩水（PBS）で乾燥したザイモサン20mgを加えます。 10分間沸騰水浴中で渦と熱。クールで5分間2,000×gで遠心します。 </li><li>水浴ソニケーターで、上清を除去し1mlのPBS中に再懸濁し、10分間超音波処理。 2 1.5ミリリットルチューブに500μlのを転送します。 5分間11,000×gで遠心分離します。 </li><li> 500μlのPBSで洗浄を繰り返します。煮沸ザイモサンは、等分凍結し、-20℃で保存することができます。 </li><li>たて、0.1M の Na 2 CO 3、（ステップ1.2）上記のようにpHが9.3を用意し500μlの二回ザイモサンを洗ってください。 </li><li>最終洗浄後490μlの再懸濁。ジメチルsulfoxidに溶解し10 mg / mlのFITCの10μLを追加E（DMSO）、ボルテックス、ホイルで覆い、そして室温で4時間撹拌しながらインキュベートします。 </li><li>最初の0.5ミリリットルのDMSO +300μlのPBS、その後、0.5ミリリットルのDMSO +450μlのPBS、その後、PBSのみで、その後+150μlのPBS 0.5ミリリットルのDMSOで、上記のように（ステップ1.2）洗浄し、結合していない色素を除去するために。 </li><li>血球計数器を使用してザイモサンを数えます。それは中央のグリッド上に配置されたカバースリップを満たしている血球計数器室の端に10μlを添加、ボルテックス、500μlのPBSにザイモサンの1μlを添加します。 20Xの対物レンズを搭載した明視野顕微鏡で血球計数器を取り付けます。 </li><li> 16正方形を含む左上隅に焦点を当て、手の数取器を使用して、このエリア内のすべてのザイモサン粒子をカウントします。右下の隅に繰り返します。 =ザイモサン濃度/ 2×500×10 4ザイモサン/ mlのカウント：以下の式を使用してザイモサン濃度を計算します。標識ザイモサンは、等分凍結し、-20℃で保存することができます。 </li><li>ラベルされたザイモサンのwiをオプソニン化1：100希釈でウサギ抗ザイモサン抗体番目。ボルテックスおよび37℃の水浴中で1時間インキュベートします。 （ステップ1.2）上記のように500μlのPBSで洗浄します。 注意：ザイモサン後で解析をより困難にすることができ塊を形成する傾向があるとして、超音波処理は非常に、特にオプソニン化した後に、推奨されます。 65％（v / v）のラットまたはヒト血清でオプソニン化は、代替として使用することができます。 </li><li>ステップ1.7から1.8に記載されているように血球計数器を使用してザイモサンをカウントし、100×10 6粒子/ mlに希釈します。補体、または全くオプソニンのような他の食細胞刺激剤とのオプソニン化は、代わりに実行することができます。 </li></ol> 2.ライブビデオ顕微鏡<ol><li>他の場所38で詳細に説明するように、一次マウスの好中球を分離します。 <ol><li>また、任意の食細胞タイプを使用します。 （RPMI）ロズウェルパーク記念研究所で氷の上での好中球を保ち、5％ウシ胎児血清を補充した1640（V / V）、200 Uペニシリン/ streptomycinは、10×10 6細胞/ mlの濃度で使用直前まで。他の細胞型は、1〜4日前に播種することができます。 </li></ol></li><li> 50μlの培地を追加することで、ドロップを広げ、細胞を接着させるために37℃で5分間インキュベートし、35mmのガラスボトムディッシュの中心に好中球の2×10 5（20μl）を追加します。 </li><li>ハンクス平衡塩溶液（HBSS）1mlを特定のMPO阻害剤、4-アミノ安息香酸ヒドラジド（4- ABH、10μM）を含む37℃に温め加えます。このようなアジ化ナトリウムのような非特異的なMPO阻害剤、（5 mM）を、使用してもよいが、近年pHを39に追加の非特異的効果を発揮することが示唆されています。 </li><li> 37℃の加熱システムと40X油目的を搭載した広視野蛍光顕微鏡の料理をマウントします。細胞や機器を平衡化することを可能にするために10分間イメージングせずに細胞をインキュベートします。 </li><li>明視野透過画像を取得する顕微鏡の設定を調整する[PHASEまたは440 nm以下490nmの励起および535nm放射ごとに30秒で使用可能な場合は微分干渉コントラスト（DIC）]。 </li><li>あまりにも多くの光退色や光毒性を回避するために、顕微鏡システムに応じてと頼まれて、実験の質問に応じて露光時間と取得頻度を調整します。画像取得を開始します。 </li><li> 2分後、10×10 6（50μl）を追加皿の中央にFITC-ザイモサンをオプソニン化。使用食細胞とターゲットの種類に応じて細胞比：ターゲットを調整します。 </li><li> 30分間撮影を続けます。 30分後、タイムラプス取得を停止し、タイマーを開始します。ステージを移動し、すべての5分以内に、画像その他のファゴソームに異なる視野で10以上のスナップショットをキャプチャします。 </li></ol> 3.キャリブレーションとコントロール実験<ol><li>実験の日に（レシピは表1に記載されている）は、従来のpH校正ソリューションを準備し、10mlのアリコートで凍結します。37℃の水浴中に較正溶液と暖かいを解凍します。 pHメーターでpHをチェックして、ソリューションの実際のpHを記録します。 </li><li>前の画像取得にガラスボトムディッシュの蠕動ポンプをマウントして、上記のようにライブビデオ顕微鏡を行う（セクション2）。 </li><li> 30分の取得期間の終わりに、ディッシュ内の溶液を除去第1の較正溶液1mlを加え、ポンプを停止する上でポンプの電源を入れます。 </li><li>信号は、通常は5分を安定するまで待ちます。各校正液で繰り返します。 <ol><li>実験日ごとに少なくとも1つのキャリブレーションを実行して、最高のpHで始まり、最低に順次作業だけでなく、最低のpHから出発して、最高に向けて順次作業を調整します。 </li></ol></li><li>対照として、ガラスボトムディッシュで900μlのHBSS中の細胞にHBSS中で希釈し、100μMNADPHオキシダーゼ阻害剤ジフェニルpropioniumヨウ化（DPI）の100μlを添加し、10分間インキュベートします。 </li><li>取得を開始して（ステップ2.7）上記のようにザイモサンを追加します。食作用の15分後、HBSS中で希釈した（コンカ）コンカナマイシン、10μMのV-ATPaseの阻害剤の10μlを添加し、さらに15分間撮影を続けます。 </li></ol> 4.分析<ol><li>タイムラプス映画やファゴソームスナップショットの分析。 注意：以下の手順は、ImageJのに特異的です。ステップバイステップの手順は、使用するソフトウェアの依存して広く変化し得るが、この節で説明する関数は、ほとんどのプロの画像解析ソフトで利用できます。 <ol><li>プロの画像解析ソフトウェアで画像を開きます。何の細胞が存在しない領域に四角ポリゴン選択ツールを使用して、小さな正方形のROIを描画し、ROIからプラグイン&gt; BGの減算をクリックすることで、490チャンネルのバックグラウンドを減算します。次に&gt;選択&gt;選択と繰り返しを復元し、[編集]をクリックすることで、440チャンネルで同じROIをアップロードします。 </li><li>その後、[操作]ドロップダウンメニューで「分割」を選択、...プロセス&gt;画像電卓をクリック画像1と画像2のドロップダウンメニューで、適切な画像を選択することにより、440チャンネルで割った490チャンネルの32ビットの比画像を作ります、および32ビット（フロート）結果チェック・ボックスをチェック。 </li><li> &gt;レインボーRGBの画像&gt;ルックアップテーブルをクリックすることで、比と互換性の色分けにカラーコーディングルックアップテーブルを変更します。画像をクリックして、0と無限大のピクセルを排除するための閾値比画像は、&gt;&gt;しきい値を...調整します。 NaNのチェックボックスに設定した背景ピクセルが選択されていることを確認すること、そのザイモサンは赤で表示されるようにスクロールバーを調整し、[適用]をクリックします。 </li><li>画像をクリックすることで、マルチチャンネルコンポジットに明視野チャンネルとこの比スタックを組み合わせ&gt;カラー&gt;チャンネルをマージし、灰色のチャネルのドロップダウンメニューで、明視野像と緑のチャネルのドロップダウンメニューで、比画像を選択し、選択しますチェックボックスをソース画像を保管してください。タイムラプスムービーやファゴソームが分析されるかを決定するためにスナップショットをスキャンします。明視野チャンネルは、この場合に便利です。 </li><li>追加&gt;ファゴソームの周りに楕円形、多角形選択ツールを使用して、関心領域（ROI）を描画し、分析]&gt; [ツール]&gt; [ROIマネージャ]をクリックして、ROIマネージャーに追加します。詳細&gt;マルチ測定デスライス選択]チェックボックスごとに1つの行をクリックすることで、彼らのザイモサン強度比を測定します。 </li><li>タイムラプスムービーでは、トラックは、各時点を測定するには、Ctrl + Mを入力し、ROIを描画し、時間をかけて強度値を追跡するために必要な場合に、ROIを移動させることにより、一度に一つのファゴソーム。表計算ソフトに結果ウィンドウからの測定値をコピーします。 注：15（実験X 3当たり5時間のコース）の独立した実験の分析は理想的ですが、多かれ少なかれ観察変動に応じて必要となる場合があります。 ROIを保存すると、常に推奨されます。一部のソフトウェアパッケージでは、画像レジを許可ストレーションおよびROIの動的更新、分析のこの部分を容易にします。 </li></ol></li><li>蛍光のpH値への変換<ol><li> 4.1節で説明したように、キャリブレーションタイムラプスムービーでファゴソームのための440分の490の比率を測定します。 </li><li>スプレッドシートでは、時間をかけて比の​​値をプロットし、各pH校正に対応する期間の中央内5の時点から（5-20​​の間、通常は）視野内の全てのファゴソームから平均の比の値を計算します溶液。 （ 図5Aにも赤いボックスを参照してください。） </li><li>実際に測定されたpH値に対してこれらの平均440分の490比の値をプロットします。一つのグラフに、少なくとも3つの独立したキャリブレーションを結合します。通常シグモイド方程式に、非線形回帰（ベストフィット曲線）を実行するために、統計や数値ソフトウェアパッケージを使用してください。 注：例えば、 図5（b）で使用される式は、ボルツマンシグモイド[Y =ボトム+（（上下）でした/（1 + EXP（（V50-X）/スロープ））Yの値が440分の490比である]、Xは、pHの値であり、上、下、V50及び傾斜パラメータは、ソフトウェアによって計算されます。 </li><li> pHに比の値を変換するために取得した式を使用します。 注：例えば、 図5（b）にアジ化ナトリウムの存在下での検量線について得られた方程式は440分の490率で= 1.103 + [（2.89から1.103）/（1 + EXP（（6.993-PH）/0.9291）] 。pHを解くことは、以下の式が得られます。pH値= 6.993から0.9291 * [LN（（1.178 /（比-1.103）） - 1））]。 </li></ol></li></ol>

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virulent+and+avirulent+strains+of+Francisella+tularensis+prevent+acidification+and+maturation+of+their+phagosomes+and+escape+into+the+cytoplasm+in+human+macrophages.">Virulent and avirulent strains of Francisella tularensis prevent acidification and maturation of their phagosomes and escape into the cytoplasm in human macrophages.</a> Infect Immun. 72 (6), 3204-3217 (2004).</li><li>Alpuche-Aranda, C. M., Swanson, J. A., Loomis, W. P., Miller, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+typhimurium+activates+virulence+gene+transcription+within+acidified+macrophage+phagosomes.">Salmonella typhimurium activates virulence gene transcription within acidified macrophage phagosomes.</a> Proc Natl Acad Sci U S A. 89 (21), 10079-10083 (1992).</li><li>Lukacs, G. L., Rotstein, O. D., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+the+phagosomal+pH+in+macrophages.+In+situ+assessment+of+vacuolar+H(+)-ATPase+activity,+counterion+conductance,+and+H++'leak'.">Determinants of the phagosomal pH in macrophages. In situ assessment of vacuolar H(+)-ATPase activity, counterion conductance, and H+ 'leak'.</a> J Biol Chem. 266 (36), 24540-24548 (1991).</li><li>Watanabe, K., Kagaya, K., Yamada, T., Fukazawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+for+candidacidal+activity+in+macrophages+activated+by+recombinant+gamma+interferon.">Mechanism for candidacidal activity in macrophages activated by recombinant gamma interferon.</a> Infect Immun. 59 (2), 521-528 (1991).</li><li>Ip, W. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis+and+phagosome+acidification+are+required+for+pathogen+processing+and+MyD88-dependent+responses+to+Staphylococcus+aureus.">Phagocytosis and phagosome acidification are required for pathogen processing and MyD88-dependent responses to Staphylococcus aureus.</a> J Immunol. 184 (12), 7071-7081 (2010).</li><li>Clarke, M., Maddera, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocyte+meets+prey:+uptake,+internalization,+and+killing+of+bacteria+by+Dictyostelium+amoebae.">Phagocyte meets prey: uptake, internalization, and killing of bacteria by Dictyostelium amoebae.</a> Eur J Cell Biol. 85 (9-10), 1001-1010 (2006).</li><li>Jankowski, A., Scott, C. C., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+the+phagosomal+pH+in+neutrophils.">Determinants of the phagosomal pH in neutrophils.</a> J Biol Chem. 277 (8), 6059-6066 (2002).</li><li>Segal, A. W., Geisow, M., Garcia, R., Harper, A., Miller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+respiratory+burst+of+phagocytic+cells+is+associated+with+a+rise+in+vacuolar+pH.">The respiratory burst of phagocytic cells is associated with a rise in vacuolar pH.</a> Nature. 290 (5805), 406-409 (1981).</li><li>Savina, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NOX2+controls+phagosomal+pH+to+regulate+antigen+processing+during+crosspresentation+by+dendritic+cells.">NOX2 controls phagosomal pH to regulate antigen processing during crosspresentation by dendritic cells.</a> Cell. 126 (1), 205-218 (2006).</li><li>Rybicka, J. M., Balce, D. R., Chaudhuri, S., Allan, E. R., Yates, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagosomal+proteolysis+in+dendritic+cells+is+modulated+by+NADPH+oxidase+in+a+pH-independent+manner.">Phagosomal proteolysis in dendritic cells is modulated by NADPH oxidase in a pH-independent manner.</a> EMBO J. 31 (4), 932-944 (2012).</li><li>Mantegazza, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NADPH+oxidase+controls+phagosomal+pH+and+antigen+cross-presentation+in+human+dendritic+cells.">NADPH oxidase controls phagosomal pH and antigen cross-presentation in human dendritic cells.</a> Blood. 112 (12), 4712-4722 (2008).</li><li>Balce, D. R., Allan, E. R., McKenna, N., Yates, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gamma-Interferon-inducible+lysosomal+thiol+reductase+(GILT)+maintains+phagosomal+proteolysis+in+alternatively+activated+macrophages.">gamma-Interferon-inducible lysosomal thiol reductase (GILT) maintains phagosomal proteolysis in alternatively activated macrophages.</a> J Biol Chem. 289 (46), 31891-31904 (2014).</li><li>Sanjurjo, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+scavenger+protein+apoptosis+inhibitor+of+macrophages+(AIM)+potentiates+the+antimicrobial+response+against+Mycobacterium+tuberculosis+by+enhancing+autophagy.">The scavenger protein apoptosis inhibitor of macrophages (AIM) potentiates the antimicrobial response against Mycobacterium tuberculosis by enhancing autophagy.</a> PLoS One. 8 (11), e79670 (2013).</li><li>Yuan, Z., Zhi, L., Li-Juan, Z., Hong-Tao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Utility+of+Chloroquine+in+Cancer+Therapy.">The Utility of Chloroquine in Cancer Therapy.</a> Curr Med Res Opin. , 1-12 (2015).</li><li>Ohkuma, S., Poole, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+probe+measurement+of+the+intralysosomal+pH+in+living+cells+and+the+perturbation+of+pH+by+various+agents.">Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents.</a> Proc Natl Acad Sci U S A. 75 (7), 3327-3331 (1978).</li><li>El Chemaly, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VSOP/Hv1+proton+channels+sustain+calcium+entry,+neutrophil+migration,+and+superoxide+production+by+limiting+cell+depolarization+and+acidification.">VSOP/Hv1 proton channels sustain calcium entry, neutrophil migration, and superoxide production by limiting cell depolarization and acidification.</a> J Exp Med. 207 (1), 129-139 (2010).</li><li>Levine, A. P., Duchen, M. R., Segal, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+HVCN1+channel+conducts+protons+into+the+phagocytic+vacuole+of+neutrophils+to+produce+a+physiologically+alkaline+pH.">The HVCN1 channel conducts protons into the phagocytic vacuole of neutrophils to produce a physiologically alkaline pH.</a> bioRxiv. , (2014).</li><li>Al-Fageeh, M. B., Smales, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+and+regulation+of+the+cellular+responses+to+cold+shock:+the+responses+in+yeast+and+mammalian+systems.">Control and regulation of the cellular responses to cold shock: the responses in yeast and mammalian systems.</a> Biochem J. 397 (2), 247-259 (2006).</li><li>Yui, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basolateral+targeting+and+microtubule-dependent+transcytosis+of+the+aquaporin-2+water+channel.">Basolateral targeting and microtubule-dependent transcytosis of the aquaporin-2 water channel.</a> Am J Physiol Cell Physiol. 304 (1), C38-C48 (2013).</li><li>Griffiths, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+phagosome+individuality+and+membrane+signalling+networks.">On phagosome individuality and membrane signalling networks.</a> Trends Cell Biol. 14 (7), 343-351 (2004).</li><li>Poburko, D., Santo-Domingo, J., Demaurex, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+regulation+of+the+mitochondrial+proton+gradient+during+cytosolic+calcium+elevations.">Dynamic regulation of the mitochondrial proton gradient during cytosolic calcium elevations.</a> J Biol Chem. 286 (13), 11672-11684 (2011).</li></ol>

The authors have nothing to disclose.

ターゲットに接続されたpH感受性色素を用いた同様の戦略を採用するが、ファゴソームの集​​団の平均pH値を測定し、このような分光法およびFACSなどの代替の方法、より多くの時間がかかりますが、顕微鏡検査は、いくつかの利点を提供しています。最初に、内部および外部で結合したことであるが、内在化しない、粒子は容易にそれぞれ、外部粒子を急冷または標識するために、トリパ?...

Phagocytosis, the process through which innate immune cells engulf large particles, evolved from the eating mechanism of single-celled organisms, and involves binding to a target, enveloping it with a membrane and pinching the membrane off to form a vacuole within the cytosol called a phagosome. While the phagosomal membrane is derived from the plasma membrane, active protein and lipid sorting, as well as fusion with endomembranes during phagosome formation, transform the phagosome into a distinct organelle within the cell with degradative properties that allow the killing, neutralization and breakdown of the ingested material1-3. This process, called phagosomal maturation, relies on the delivery of a host of proteolytic and microbicidal enzymes, including the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase which transfers electrons into phagosomes producing the strong oxidant O2- and its derivative reactive oxygen species (ROS) 2,4. 
The luminal pH of phagosomes has a profound influence on several events required for phagosome function. First, pH influences trafficking of endomembranes in general, as pH-dependent conformational changes of transmembrane trafficking regulators alters the recruitment of trafficking determinants such as Arfs, Rabs and vesicular coat-proteins, which in turn define which vesicles may fuse with phagosomes 5-8. Second, the ionic composition of the phagosomal lumen is also transformed as phagosomes mature, and some ion transporters, such as the Na+/H+ exchanger or ClC family Cl-/H+ antiporters, which promote phagocytic function, rely on H+ translocation 9,10. Similarly, ROS production is intimately linked with phagosomal pH. ROS and its toxic oxidant byproducts have long been recognized as crucial for phagosomal killing in neutrophils 4,11,12, and have been shown to play critical roles in other phagocytes including macrophages, dendritic cells (DCs) and amoeba 13-16. The NADPH oxidase is an electrogenic enzyme that releases H+ in the cytosol as NADPH is consumed, and that requires the simultaneous transfer of H+ through companion HVCN1 channels alongside the transported electrons into the phagosomal lumen, in order to alleviate the massive depolarization that would otherwise lead to self-inhibition of the enzyme 17-21. Finally, several proteolytic enzymes have optimal activity at different pH, so time-dependent phagosomal pH changes can influence which enzymes are active and when. The importance of phagosomal pH is highlighted by organisms such as Mycobacterium tuberculosis, Franciscella tularensis and Salmonella typherium that subvert phagocytic killing at least in part by altering phagosomal pH 22-24.
In mammals the main phagocytes are neutrophils, macrophages and dendritic cells, and depending on cell type, time-dependent phagosomal pH changes can vary widely, and appear to play different roles. In macrophages a strong and rapid acidification mediated by the ATP-dependent proton pump vacuolar ATPase (V-ATPase) is one of the key factors mediating killing 25-27, resembling the mechanisms present in amoeba that use phagocytosis as an eating mechanism 28. In these cells activation of acidic proteases is thought to play a key role. In contrast, neutrophil killing relies more on ROS as well as HOCl produced by myeloperoxidase (MPO)11, and the pH remains neutral or alkaline during the first 30 min acidifying only later 29,30. Neutral pH has been suggested to favor the activity of oxidative proteases such as certain cathepsins. In DCs phagosomal pH is controversial, with some reporting acidification and others neutral or alkaline pH 31,32, but ROS and pH may profoundly influence the ability of these cells to present antigens to T cells, one of their main functions 33. 
Importantly, hormones, chemokines and cytokines may produce signaling events that induce maturation and changes in phagocyte behavior, and in turn influence phagosomal pH 34,35. Similarly, drugs, for example the antimalarial chloroquine, which is also considered for anti-cancer therapies 36, may affect phagosomal pH and therefore immune outcomes. Thus, a variety of cell biologists, immunologists, microbiologists and drug developers may be interested in measuring phagosomal pH when seeking to understand the mechanisms underlying the effects of a particular genetic disruption, bioactive compound or microorganism on innate and adaptive immune responses. 
Here we describe a method for measuring phagosomal pH in neutrophils that allowed us to show the importance of the HVCN1 channel in regulating neutrophil phagosomal pH 19. The method is based on the ratiometric property of fluorescein isothiocyanate (FITC) whose fluorescence emission at 535 nm is pH sensitive when excited at 490 nm but not 440 nm 37. When this dye is chemically coupled to a target, in this case zymosan, it can be followed using wide-field fluorescence microscopy, where cells are imaged as they phagocytose, and phagosomal pH changes are measured in real time as the phagosome matures. The actual pH is then gleaned by performing a calibration experiment where cells that have phagocytosed are exposed to solutions of different pH that contain the ionophores nigericin and monensin, that allow the rapid equilibration of the pH within phagosomes with the external solution. Ratio values are then compared to the known pH of solutions, a calibration curve is constructed by nonlinear regression and the resulting equation used to calculate pH from the ratio value.

<table>
	<tbody>
		<tr>
			<td>Zymosan A powder</td>
			<td>Sigma-Aldrich</td>
			<td>Z4250</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate</td>
			<td>Sigma-Aldrich</td>
			<td>F7250</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Anti-zymosan antibody (Zymosan A Bioparticles opsonizing reagent)</td>
			<td>Life Technologies</td>
			<td>Z2850</td>
			<td>Sigma-Aldrich O6637 is an equivalent product. Alternatively 25% serum can be used as an opsonizing reagent.</td>
		</tr>
		<tr>
			<td>4-Aminobenzoic hydrazide (4-ABH)</td>
			<td>Santa Cruz</td>
			<td>sc-204107</td>
			<td>Toxic, use gloves, various providers exist</td>
		</tr>
		<tr>
			<td>Diphenyleneiodonium chloride (DPI)</td>
			<td>Sigma-Aldrich</td>
			<td>D2926</td>
			<td>Toxic, use gloves, various providers exist</td>
		</tr>
		<tr>
			<td>Concanamycin A (ConcA)</td>
			<td>Sigma-Aldrich</td>
			<td>27689</td>
			<td>Toxic, use gloves, various providers exist</td>
		</tr>
		<tr>
			<td>Nigericin</td>
			<td>Sigma-Aldrich</td>
			<td>N7143</td>
			<td>Toxic, use gloves, various providers exist</td>
		</tr>
		<tr>
			<td>Monensin</td>
			<td>Enzo</td>
			<td>ALX-380-026-G001</td>
			<td>Toxic, use gloves, various providers exist</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Life Technologies</td>
			<td>14200-075</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Hank&#39;s balance salt solution</td>
			<td>Life Technologies</td>
			<td>14025092</td>
			<td>Ringer&#39;s balanced salt solution or other clear physiological buffers may be substituted.</td>
		</tr>
		<tr>
			<td>Sodium carbonate (Na2CO3)</td>
			<td>Sigma-Aldrich</td>
			<td>S7795</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>2-(N-Morpholino)ethanesulfonic acid (MES)</td>
			<td>Sigma-Aldrich</td>
			<td>M3671&#160;</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES)</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>N-Methyl-D-glucamine (NMDG)</td>
			<td>Sigma-Aldrich</td>
			<td>M2004&#160;</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid (EGTA)</td>
			<td>Sigma-Aldrich</td>
			<td>3777</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>&#160;Tris(hydroxymethyl)aminomethane (Tris)</td>
			<td>Sigma-Aldrich</td>
			<td>T1503&#160;</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P9333</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Magnesium chloride (MgCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Absolute Ethanol (EtOH)</td>
			<td>Sigma-Aldrich</td>
			<td>2860</td>
			<td>Various providers exist</td>
		</tr>
		<tr>
			<td>Glass-bottom 35 mm petri dishes (Fluorodish)</td>
			<td>World Precision Instruments</td>
			<td>FD35-100</td>
			<td>Ibidi &#181;-clear dishes or coverslips with appropriate imaging chambers may be sustituted</td>
		</tr>
		<tr>
			<td>Sonicating water bath</td>
			<td>O. Kleiner AG</td>
			<td></td>
			<td>A sonicator may be used instead, various instrument providers exist</td>
		</tr>
		<tr>
			<td>Heamocytometer</td>
			<td>Marienfeld GmbH</td>
			<td></td>
			<td>Various instrument providers exist</td>
		</tr>
		<tr>
			<td>Widefield live imaging microscope</td>
			<td>Carl Zeiss AG</td>
			<td></td>
			<td>Various instrument providers exist, but the microscope must be able to image 440/535 and 490/535 excitation/emission respective. Spinning disk confocal set-ups with brightfield capabilities may substituted, but zymosan tend to go out of focus more often.&#160;&#160;</td>
		</tr>
		<tr>
			<td>Peristaltic pump (Dynamax RP-1)</td>
			<td>Rainin</td>
			<td></td>
			<td>Various instrument providers exist</td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Schott Gerate GmbH</td>
			<td></td>
			<td>Various instrument providers exist</td>
		</tr>
		<tr>
			<td>Manual Counter</td>
			<td>Milian SA</td>
			<td></td>
			<td>Various instrument providers exist</td>
		</tr>
	</tbody>
</table>

measuring phagosome ph by ratiometric fluorescence microscopy

食作用は、先天性免疫細胞が死滅させるかまたは中和摂取材料を、または抗原として​​それを提示し、適応免疫応答を開始するために、細菌、アポ​​トーシス細胞または他の異物を飲み込み、それを通して基本的な過程です。ファゴソームのpHは、内膜およびタンパク質分解酵素の活性化、食作用性空胞が分解オルガネラに成熟することを可能にするイベントと核分裂や核融合を制御する重要なパラメータです。加えて、H +との転座は、他の宿主組織への効率的な殺傷及びシグナリングのために必須である活性酸素種（ROS）の高レベルの産生のために必要とされます。多くの細胞内病原体は、フ​​ァゴソームの生物学におけるpHの重要性を強調し、ファゴソーム酸性化を制限することにより、食細胞死滅を覆します。ここでは、フルオレセインイソチオシアネート（FITC）を使用して、好中球にファゴソームのpHを測定するためのレシオメトリック法は貪食TARGとしてザイモサンを標識説明しますETS、ライブセルイメージング。アッセイは、単一色素の蛍光よりもむしろ絶対的なpH依存性比の定量を、可能にする、440 nmで励起した場合、490nmで励起した場合、酸性pHによりクエンチされないが、FITCの蛍光特性に基づいています。 インサイチュ染料較正及び実際のpH値との比の変換を行うための詳細なプロトコルも提供されます。彼らは、このような漂白などの摂動に対する感度が低いなどの単一染料レシオメトリック法は、一般的に測定された信号を歪ま変化、レーザーの変動、および不均一なラベルを、集中、単一の波長またはデュアル色素擬似レシオメトリックのプロトコルよりも優れて考えられています。この方法は、容易に他の食細胞タイプのpHを測定するために変更することができ、及びザイモサンは、不活性ビーズからの生きた微生物を、任意の他のアミン含有粒子で置き換えることができます。最後に、この方法は、異なるpH範囲に感受性の他の蛍光プローブまたは他のphagosomを利用するように適合させることができますアル活動は、それファゴソームの機能イメージングのための一般的なプロトコルとなっています。

/ - -以下は、初代マウス好中球のファゴソームpHは野生型またはHvcn1の骨髄から分離された実験の代表的な結果であります マウスを比較しました。成功した実験では、それは後の画像解析の際にセグメントに、より困難になりますあまりにも多くのファゴソームを回避しながら、タイムラプスムービーの全期間中に視野内に十分なファゴソームを得ることが重要で...

Watch this Scientific Journal Video about Measuring Phagosome pH by Ratiometric Fluorescence Microscopy at JoVE.com

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy.

レシオメトリック蛍光顕微鏡によってファゴソームのpHを測定します

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or ...

measuring-phagosome-ph-by-ratiometric-fluorescence-microscopy

Research

JoVE Journal

Immunology and Infection

13.8K ビュー.  University of Geneva. Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy. 

ビデオ: Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

マイクロ波は、透過型電子顕微鏡による植物ウイルス病の迅速診断をアシスト

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

免疫・感染学

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 ...

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

ライブマクロファージにおけるファゴリソソーム生合成の研究

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

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

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

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 ...

Ratiometric Imaging of Extracellular pH in Dental Biofilms

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites are not distributed evenly in dental biofilms. A complex interplay of sorption to and reaction with organic matter in the biofilm reduces the diffusion paths of solutes and creates steep gradients of reactive molecules, including organic acids, across the biofilm. Quantitative fluorescent microscopic methods, such as fluorescence life time imaging or pH ratiometry, can be employed to visualize pH in different microenvironments of dental biofilms. pH ratiometry exploits a pH-dependent shift in the fluorescent emission of pH-sensitive dyes. Calculation of the emission ratio at two different wavelengths allows determining local pH in microscopic images, irrespective of the concentration of the dye. Contrary to microelectrodes the technique allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) is employed to monitor extracellular pH in in vivo grown dental biofilms of unknown species composition. Upon exposure to glucose the dye is up-concentrated inside all bacterial cells in the biofilms; it is thus used both as a universal bacterial stain and as a marker of extracellular pH. After confocal microscopic image acquisition, the bacterial biomass is removed from all pictures using digital image analysis software, which permits to exclusively calculate extracellular pH. pH ratiometry with the ratiometric dye is well-suited to study extracellular pH in thin biofilms of up to 75 &#181;m thickness, but is limited to the pH range between 4.5 and 7.0.

A pH-sensitive ratiometric dye is used in combination with confocal laser scanning microscopy and digital image analysis to monitor extracellular pH in dental biofilms in real-time.

歯科バイオフィルムにおける細胞外のpHのレシオメトリックイメージング

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites ...

"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 ...

Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular compartment where the killing and digestion of engulfed particles take place, is the main arena for neutrophil pathogen killing that requires tight regulation. Phagosomal pH is one aspect that is carefully controlled, in turn regulating antimicrobial protease activity. Many fluorescent pH-sensitive dyes have been used to visualize the phagosomal environment. S-1 has several advantages over other pH-sensitive dyes, including its dual emission spectra, its resistance to photo-bleaching, and its high pKa. Using this method, we have demonstrated that the neutrophil phagosome is unusually alkaline in comparison to other phagocytes. By using different biochemical conjugations of the dye, the phagosome can be delineated from the cytoplasm so that changes in the size and shape of the phagosome can be assessed. This allows for further monitoring of ionic movement.

This manuscript describes a simple method to measure the phagosomal pH and area as well as the cytoplasmic pH of human and mouse neutrophils using the ratiometric indicator seminaphthorhodafluor (SNARF)-1, or S-1. This is achieved using live-cell confocal fluorescence microscopy and image analysis.

レシオメトリックpH指示薬を使用して好中球ファゴソームと細胞質のイメージング

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular ...

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

蛍光寿命イメージングによる細胞内のスネアを可視化

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...

Measuring Deformability and Red Cell Heterogeneity in Blood by Ektacytometry

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of disease or occurrence of serious complications. Ektacytometry uses laser diffraction viscometry to measure the deformability of red blood cells subject to either increasing shear stress or an osmotic gradient at a constant value of applied shear stress. However, direct deformability measurements are difficult to interpret when measuring heterogenous blood that is characterized by the presence of both rigid and deformable red cells. This is due to the inability of rigid cells to properly align in response to shear stress and results in a distorted diffraction pattern marked by an exaggerated decrease in apparent deformability. Measurement of the degree of distortion provides an indicator of the heterogeneity of the erythrocytes in blood. In sickle cell anemia, this is correlated with the percentage of rigid cells, which reflects the hemoglobin concentration and hemoglobin composition of the erythrocytes. In addition to measuring deformability, osmotic gradient ektacytometry provides information about the osmotic fragility and hydration status of erythrocytes. These parameters also reflect the hemoglobin composition of red blood cells from sickle cell patients. Ektacytometry measures deformability in populations of red cells and does not, therefore, provide information on the deformability or mechanical properties of individual erythrocytes. Regardless, the goal of the techniques described herein is to provide a convenient and reliable method for measuring the deformability and cellular heterogeneity of blood. These techniques may be useful for monitoring temporal changes, as well as disease progression and response to therapeutic intervention in several disorders. Sickle cell anemia is one well-characterized example. Other potential disorders where measurements of red cell deformability and/or heterogeneity are of interest include blood storage, diabetes, Plasmodium infection, iron deficiency, and the hemolytic anemias due to membrane defects.

Here we present techniques to measure red cell deformability and cellular heterogeneity by ektacytometry. These techniques are applicable to general investigations of red cell deformability and specific investigations of blood diseases characterized by the presence of both rigid and deformable red cells in circulation, such as sickle cell anemia.

変形能と赤血球による血液中赤血球の不均一性を測定

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of ...

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with histones and antimicrobial proteins. Excessive NET formation (NETosis) and citH3 release during sepsis is associated with multiple organ dysfunction and mortality in mice and humans but its implications in dogs are unknown. Herein, we describe a technique to isolate canine neutrophils from whole blood for observation and quantification of NETosis. Leukocyte-rich plasma, generated by dextran sedimentation, is separated by commercially available density gradient separation media and granulocytes collected for cell count and viability testing. To observe real-time NETosis in live neutrophils, cell permeant and cell impermeant fluorescent nucleic acid stains are added to neutrophils activated either by lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Changes in nuclear morphology and NET formation are observed over time by fluorescence microscopy. In vitro NETosis is further characterized by co-colocalization of cell-free DNA (cfDNA), myeloperoxidase (MPO) and citrullinated histone H3 (citH3) using a modified double-immunolabelling protocol. To objectively quantify NET formation and citH3 expression using fluorescence microscopy, NETs and citH3-positive cells are quantified in a blinded manner using available software. This technique is a specific assay to evaluate the in vitro capacity of canine neutrophils to undergo NETosis.

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

We describe methods to isolate canine neutrophils from whole blood and visualize NET formation in live neutrophils using fluorescence microscopy. Also described are protocols to quantify NET formation and citrullinated histone H3 (citH3) expression using immunofluorescence microscopy.

体外蛍光顕微鏡による犬の好中球細胞外トラップ形成: ダイナミックな定量的分析

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with ...