We thank Dr Jamil Jubrail for reading the manuscript. This work was supported by grants from CNRS, Inserm, Universit&#233; Paris Descartes, Agence Nationale de la Recherche (2011 BSV3 025 02), Fondation pour la Recherche M&#233;dicale (FRM DEQ20130326518 including a doctoral fellowship for GLB) and Agence Nationale de Recherche sur le SIDA et les h&#233;patites virales (ANRS, including a post-doctoral fellowship for CD) to FN. A. Dumas was supported by doctoral fellowships from Universit&#233; Paris Descartes and Sidaction.

プロトコルは、国内および国際的な法律や地方条例に厳密に従って行わなければなりません。研究目的のために献血をする彼らの同意を与えた健康なドナーからの血液は、機関が契約を締結していると輸血センターから得られています。ヒトの血液を使用した場合には、特別な保護がとられなければなりません。 HIV-1を用いた実験では現地の法律に従って、バイオセーフティーレベル3または2（BSL-3または2）実験室で行われなければなりません。 接着によって密度勾配遠心と選択によるヒト単球由来マクロファージ（hMDMs）の調製<ol><li>健康なドナー（9ミリリットル）からの新鮮な血液で起動します。 70 mlの最終容積を得ることが2+ の Ca 2+およびMgなしの滅菌1×リン酸緩衝生理食塩水（PBS）を用いて、新鮮な血液の全体積を希釈し、穏やかに（50ml×2回）コニカルチューブ（チューブにつき35 mL）に希釈された血液を追加、の15ミリリットルの上eutralは、非常に各チューブに既に​​溶液中に、高質量の親水性多糖分岐します。 </li><li>ブレーキなしで20℃で20分間、537×gでの血液の遠心分離は、両方の管。次に界面で濁ったセル環に ​​含まれる末梢血単核細胞（PBMC）を収集およびCa 2+及びMg 2+なしで、1×PBS 15 mlを含む新しい50mlチューブに移します。 </li><li>遠心分離機20℃で5分間、218×gで細胞およびCa 2+およびMgなしで1×PBSの45ミリリットル中にペレットを再懸濁2+。 </li><li> 20℃で5分間、218×gで遠心分離した細胞は、2+ の Ca 2+およびMgなしで1×PBS 10mlにペレットを再懸濁し、トリパンブルーで1/200の最終希釈に希釈することによって細胞をカウントします。 </li><li> 20℃で5分間、218×gで細胞を遠心し、1640培地は、2mMのL-グルタミンおよび100μg/ mlのpenicを補充したRPMI（ロズウェルパーク記念研究所）でペレットを再懸濁illin -ストレプトマイシン6ウェルプレートの各ウェルに培地2ml中、ウェルあたり7×10 6個のPBMCを有すること。 </li><li> 2時間、5％CO 2、37℃でプレートをインキュベートします。 2時間後、単球をプラスチックに付着しているでしょう。 </li><li>新たに単離した単球が、hMDMsに分化培地を吸引し、2mlでhMDM培地に交換できるようにする（RPMI 1640、10％ウシ胎児血清（FCS）、2mM L-グルタミンおよび1％ペニシリン - ストレプトマイシンをdecomplemented）を補充し組換えヒトマクロファージコロニー刺激因子（RHM-CSF）を10ng / mlの最終濃度で。 </li><li> 11日目（ 図1）、5％CO 2で37℃で細胞をインキュベートします。 </li><li> 11日目で培地を除去し、ウェルあたり冷たいhMDM培地2mlで2回洗浄します。次の冷1×PBS 1mlで各ウェルを2回洗浄します。 </li><li> （2 mMのethylenediaminetetraacetatic酸と冷たい1×PBSの1ミリリットルで、各ウェル1時間を洗って、完全に分化しhMDMsを切り離すにはEDTA）と4℃で15から60分間、ウェルあたり2 mMのEDTA冷1×PBSの2ミリリットルで細胞をインキュベートします。 注：細胞が存在して剥離する別の方法（ 例えば、トリプシンまたはトリプシン様活性）良い結果11を与えるかもしれません。 </li><li> （上下十分で静かにピペッティングすることにより完成）剥離した後、細胞を回収し、冷hMDM培地10mlを含む氷上で50mlチューブに入れ。 </li><li> 20℃で5分間、218×gで遠心分離した細胞は、冷たいhMDM培地10mlにペレットを再懸濁し、トリパンブルーで1/20に希釈することによって細胞をカウントします。 </li><li> 35ミリメートル顕微鏡グレードのガラスボトムディッシュあたり1×10 6 hMDMsで細胞をシードし、1日間、5％CO 2で37℃で培養します。 </li></ol> HIV-1ウイルスストックの2生産および定量注：M. SchindlからR5向性エンベロープを運ぶNLR4.3 HIV-1のGag-iGFP（緑色蛍光タンパク質）、ギフトER 10は、マクロファージを感染させるために、リアルタイムで感染細胞を見るために使用されます。 <ol><li>商業トランスフェクション試薬を用いて対応するプロウイルスDNAの6μgの持つヒト胚性腎臓293T細胞（2×10 6 100 mmディッシュ）のトランスフェクションによりウイルスストックを生成します。 </li><li>細胞計数のβ-ガラクトシダーゼ着色が続くウイルスストックの連続希釈液を用いて、（HIV-1 LTRの制御下でβ-ガラクトシダーゼ遺伝子を有する）HeLa細胞TZM-BL指標細胞を用いたウイルスストックの感染力を定量化12青色の細胞の。 </li></ol> HIV-1とhMDMs 3.感染<ol><li> hMDMs（1.13でプレートした細胞）に感染多重度（MOI）hMDM培地の1ミリリットル中に0.02から0.03でウイルスを追加します。ウェルはhMDM媒体の唯一の1ミリリットルを追加制御し、2日間（ 図1）、5％CO 2、37℃で皿をインキュベートします。 </li><li> 2日目にhMDM MEDで細胞を洗浄イウム3回と皿当たり新鮮hMDM培地の1ミリリットルを追加します。 6日目（ 図1）、5％CO 2で37℃で細胞をインキュベートします。 </li></ol>ヒツジ赤血球の4オプソニン<ol><li>皿当たり7×10 6 SRBCをの調製のために、4分間600×gで遠心分離すると、1×PBS中の0.1％ウシ血清アルブミン（BSA）を含む溶液100μlにSRBCを2回洗浄します。 </li><li> SRBCを5μlの当たりのサブ凝集濃度でウサギのIgG抗SRBCを含むPBS / BSA 0.1％、1×500μlの洗浄SRBCをを再懸濁し、室温で30分間回転させながらインキュベートします。 注：、抗SRBCをIgGのサブ凝集濃度を決定し、96ウェルプレート中の20μlの1/50から1/25600へのIgGの連続希釈物（13.1 mg / mlとでストック濃度）を調製しました。各ウェル中の20μlの2×10 6 SRBCを追加し、いくつかの時間の間、暗い部屋でプレートを置きます。サブ凝集濃度であります直前よく凝集抗体（IgG + SRBCを、ネットワークを形成する）とウェルの希釈。 </li><li>回転の後、4分間600×gでのIgGオプソニン化-SRBCをを遠心分離し、4分間、600×gの遠心分離で1×PBS / BSA 0.1％の100μlで洗います。 </li><li> 2mMのL-グルタミンおよび1％ペニシリン - ストレプトマイシン（1ミリリットル/皿）を補充した予め温めたフェノールレッドを含まないRPMI培地中のIgGオプソニン化-SRBCを再懸濁。 </li></ol> 5.ライブセルビデオ顕微鏡食作用アッセイ<ol><li>例えばCO 2は、加湿のために水でボトルを通過し、37℃で加熱室を備えた回転ディスクシステムのような共焦点イメージングシステムを使用してください。 </li><li>食作用アッセイの開始前に37℃で顕微鏡ステージを持つように、実験前に加熱室をオンにします。顕微鏡とコンピュータの電源を入れ、およびイメージングソフトウェアをロードします。 </li><li>このような走査速度、マニフィカ等の撮像設定を最適化イオン、解像度等が 60〜120分の間、毎分ごとにフィールドと、一つのフレーム画像に、少なくとも一つのセルを持っています。 注：ここでは、サンプルは63Xレンズと60分の間に1フレーム分毎に結像されます。 </li><li>顕微鏡ステージ上のイメージング皿を置きます。フィールドに一つだけの全体のHIV-1感染したマクロファージを見つけるために、フォーカスと位置を調整します。使用される撮像システムとプローブに基づいて適切な励起/発光の設定を使用します。ファゴソームを観察する明視野（BF）のチャンネルを含める（ 図1II）。透過光及び露光時間の割合を調整することにより、異なるチャネルの画像の外観を最適化します。 注：ここでは、NLR4.3 HIV-1のGag-iGFPは、レーザの20％（ 図1I）で長時間露光の50ミリ秒と491 nmのレーザーを使用して興奮していました。 </li><li>イメージング皿を外し、皿（ 図1）に7×10 6 SRBCを/ mlでSRBC懸濁液1mlを追加します。 </li><li> 500×gで遠心分離RTで2分間は、食作用を同期この遠心分離の終わりに時間を記録し、ステージに皿を返します。 </li><li>フォーカスを最適化し、（0.3ミクロンのステップ距離とセルの厚さを通して - 通常20面）のZスタックにGFPとBF画像を取り込む少なくとも1時間、毎分。使用される撮像システムのネイティブファイル形式でタイムラプスビデオを保存します。 注：ここでは、タイムラプス動画は、ネイティブ形式、* .STKファイルに保存されていました。 </li></ol>タイムラプスビデオの6.分析<ol><li>ビデオ編集ソフトの「レビュー多次元データ」ビデオ編集のために、ドロップダウンメニューをクリックして「アプリ」タブ上。 <ol><li>ファイルを開くには、「ディレクトリの選択」にし、「ベースファイルを選択」をクリックしてください。 「データ設定」ボックスで、（.nd形式）を分析し、「表示」をクリックして取得を選択します。データが列ANに時間を持つテーブルで表現されています行中のd Z-計画。 </li><li> Z-突起（ 図2、左パネル）に感染を表すために、「波長」ボックスに491 nmの波長を選択し、「すべての面」と「Z投影」タブをクリックします。 </li><li> （、時系列を分析する「波長」ボックスにBF波長を選択して、外部のSRBCを（ 図2、赤矢印）、内部SRBCを（ 図2、赤い円）と核を区別するために、Z軸上での最適な平面を選択するには図2、青丸）。 </li><li>ビデオモンタージュを保存するには、「選択[Xの]」タブで、次に「イメージのロード（S）」をクリックしてください。最後に、tifファイル形式で読み込んだ画像を保存し、ImageJソフトウェア上で次のそれらを開きます。 </li></ol></li><li>核の位置とBFチャネル（ 図3）で観察された異なるファゴソームを測定するためにImageJソフトウェア上のプラグイン「マニュアルトラッキング&quot;を使用してください。 <ol><li>ダウImageJのウェブサイトの「マニュアルトラッキング」プラグインをnload。 ImageJのでは、解析すべきプラグインと画像シーケンス（ 図3、ステップ1および2）を開きます。 </li><li>このような隣接するフレーム間の時間の量を表す「時間間隔」、および画素あたりの距離を表す「X / Yのキャリブレーション」（ 図3、ステップ3）などの設定を入力します。 注：6.45のx 6.45μmの2のピクセルサイズを持つ63Xズームとカメラが使用されているので、ここでは、各フレームと0.205ミクロンのX / Yのキャリブレーションの間に1分の時間間隔で画像シーケンスを保存します。 </li><li>追跡を開始するには、「トラックを追加」（ 図3、ステップ4）をクリックして、それが内在化される最初の時間でSRBCの中心をクリックしてください。次のフレームが自動的に表示されます。 </li><li>時間の間に位置が異なるために、すべてのフレームでSRBC中央をクリックし続けます（ 図3、赤いボックスのステップ6）。 <ol><li>その中心をクリックして、すべてのフレームでの地位を持っている核を追跡するために開始します。次回SRBCをの関数で異なるそれらの内在化（フレーム）で1ずつ（その中心をクリックして）ファゴソームを追跡します。 BFチャネルでSRBCを参照するには便宜上、トラッキング（ 図3、ステップ5）の間に「明るさとコントラスト」ウィンドウを使用します。 </li></ol></li><li>各SRBC追跡の間に、新しいトラックを持っている（ 図3、ステップ4）、「トラックを追加」をクリックしてください。追跡の数が第2の列に変更されます、結果テーブルの「トラックのn°」（ 図3、ステップ6）。 </li><li>分析の次のステップに進み、スプレッドシート内のデータを保存します。 </li></ol></li><li> （核とSRBCをの内在化後の最初の5分の間にファゴソームの速度に向かってSRBCを含有ファゴソームの走行距離を計算するためFiを提供して表計算ソフトを使用して、グレ4）。 <ol><li>スプレッドシートテーブルと新しいスプレッドシートファイルを開きます。この新たな次のパラメータファイルのみ、時間、x軸に移し、核およびすべてのSRBCを（ 図4A）のy座標。 </li><li> SRBCをのみそれらの座標と核の間の距離を計算するために、核とXY座標平面（ 図4B）でSRBCを検討してください。 注：2点間の距離は、それらを接続する経路の長さです。平面では、SRBCと核との間の距離は、ピタゴラスの定理により与えられます。 <ol><li> C（核とSRBCとの間の距離）を斜辺とAの長さを示し、B、他の2辺の長さを表す場合は、ピタゴラスの式のようにピタゴラスの定理を表現します： <img alt="式（1）" src="/files/ftp_upload/54568/54568eq1.jpg" /> 注：同時に、正規直交上、水平距離（X 核 -x SRBC）であり、垂直距離bは （SRBC -y Y 核 ）です。 </li><li>このように、核とSRBC（ 図4A、紫色のボックス）によるとの間の距離を計算します。 <img alt="式（2）" src="/files/ftp_upload/54568/54568eq2.jpg" /> 注：μm単位の距離を持っているのx / yのキャリブレーションによって（ピクセル単位）得られた距離値を乗算します。ここで、X / Yのキャリブレーションは0.205μmです。 </li><li>各時点で、核および初期距離（ 図4C）にSRBCとの間の距離を引きます。 </li><li>最初の5分間の時間に対する測定値をプロットします。プロットに線形トレンドライン（ 図5A）を適用し、SRBC内在化後の最初の5分の間に核に向かってファゴソーム速度を表す線形トレンドラインの傾きを決定します。 </li><li>の平均値と統計誤差を計算するためにデータを照合速度値と適切なソフトウェア（ 図5B）を使用して、適切な形式でそれらをプロットします。 </li></ol></li></ol></li></ol>

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The authors have nothing to disclose.

この技術は、いくつかの重要なステップがあります。感染の割合がドナー依存しているため、まず、HIV-1によるhMDMsの製造およびそれらの感染が重要です。注目すべきは、我々は潜在的に生体内でウイルスが遭遇したマクロファージの状態がよく、これまでに特徴付けされていないため、感染前に、in vitroで偏光されていないマクロファージを使用することを決定しました。我々?...

Macrophages play a major role in the innate immune system and in homeostasis. They are professional phagocytes that internalize and eliminate pathogens and debris by a process called phagocytosis 1,2. The phagosome, the closed compartment that forms after the engulfment of particulate material, undergoes a series of fusion and fission events with endocytic compartments, leading to a degradative compartment called the phagolysosome. This compartment has an acidic pH, due to the acquisition of proton-pumping v-ATPases, contains hydrolytic enzymes and is enriched in lysosomal-associated membrane proteins (LAMPs). The maturation of phagosomes is accompanied by their migration on microtubules 3,4 towards the cell center to reach a perinuclear location where lysosomes are accumulated.
Many pathogens have been reported to hijack phagosome maturation, including bacteria with intracellular lifestyles that modify the vacuolar environment where they reside 5. The Human Immunodeficiency Virus (HIV)-1 targets macrophages in addition to T cells. As macrophages are resistant to the cytopathic effects of the virus, unlike T cells, they can be considered as a reservoir for the virus. In addition, macrophages infected with HIV-1 show defective phagocytic functions and contribute to the emergence of opportunistic diseases. In particular, severe invasive non-typhoidal Salmonella disease caused by Salmonella Typhimurium ST313 has been prevalent for the last three decades in sub-Saharan African children or adults infected with HIV 6. It has been estimated that the risk of developing tuberculosis is more than 20 times greater in people living with HIV than among those without HIV infection. 
For all these reasons, it is important to better define the molecular mechanisms underlying the phagocytic defects in HIV-infected macrophages. We have shown that the uptake of particulate material, opsonized particles, bacteria or fungi, was inhibited in HIV-infected macrophages 7. Given that this inhibition is partial, we then set out to analyze the fate of the internalized phagosomes in HIV infected human macrophages 8. Because phagosome maturation is tightly connected with migration to the cell center and fusion with lysosomes, a defect in phagosomal maturation can be due to modifications of the trafficking modalities in the infected cell. The method described here uses IgG-opsonized Sheep Red Blood Cells (IgG-SRBCs) as a model to target receptor-mediated phagocytosis and in particular receptors for the Fc portion of immunoglobulins (FcR). These particles are easier to image in bright field (BF) than latex beads because extracellular and intracellular SRBCs show different refraction properties 9. To measure the velocity of phagosomes moving towards the nucleus in HIV-infected macrophages, we used a fluorescent virus 10 and set up a simple manual tracking method that is described here. The method does not require advanced programming and simply uses ImageJ. It is amenable to adherent cells and any type of particle or pathogen that can be visualized with bright field microscopy or with fluorescent imaging.

<table><tbody><tr><td>Falcon 100 mm TC-Treated Cell Culture Dish</td><td>Corning</td><td>353003</td><td>For viral production</td></tr><tr><td>Glass Bottom Dishes 35 mm uncoated 1.5</td><td>MatTek corporation</td><td>P35G-1.5-14-C Case</td><td>For acquisition</td></tr><tr><td>Falcon Tissue Culture Plates 6-well</td><td>Thermo Fischer Scientific</td><td>Corning. Inc. 353934</td><td>For human monocyte-derived macrophages</td></tr><tr><td>Ficoll-Plaque PLUS</td><td>Dominique Dutscher</td><td>17-1440-03</td><td>a neutral, highly branched, high-mass, hydrophilic polysaccharide in solution for density centrifugation</td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>Thermo Fischer Scientific</td><td>14190-094</td><td>RT</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM) 1x, liquid (High Glucose)</td><td>GIBCO, Molecular probes</td><td>31966-021</td><td>Conserved at 4 &amp;deg;C ; for HEK cells culture</td></tr><tr><td>RPMI 1640 medium GLUTAMAX&amp;nbsp; Supplement</td><td>Life technologies</td><td>61870-010</td><td>Conserved at 4 &amp;deg;C; for hMDMs culture</td></tr><tr><td>F&amp;oelig;tal Calf Serum (FCS)&amp;nbsp;</td><td>Eurobio</td><td>CVFSVF0001</td><td>Conserved at -20 &amp;deg;C ; decomplemented</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>Thermo Fischer Scientific</td><td>15140-122</td><td>Conserved at -20 &amp;deg;C ; for hMDMs culture</td></tr><tr><td>RPMI 1640 medium, no phenol red (10x 500 ml)</td><td>Life technologies</td><td>11835-105</td><td>Warm in 37 &amp;deg;C water bath before use ; for phagocytosis assay</td></tr><tr><td>FuGENE6 Transfection Reagent</td><td>Promega</td><td>E2692</td><td>Conserved at 4 &amp;deg;C ; for viral production</td></tr><tr><td>Sheep red blood cells (SRBCs)</td><td>Eurobio</td><td>DSGMTN00-0Q</td><td>Conserved in Alsever buffer at 4 &amp;deg;C before use</td></tr><tr><td>Anti-sheep red blood cells IgG</td><td>MP Biomedicals</td><td>55806</td><td>Conserved at 4 &amp;deg;C</td></tr><tr><td>Bovine Serum Albumin heat shock fraction, pH 7, &amp;ge;98%&amp;nbsp;</td><td>Sigma</td><td>A7906</td><td>Conserved at -20 &amp;deg;C</td></tr><tr><td>Inverted microscope DMI600</td><td>Leica</td><td></td><td></td></tr><tr><td>Confocal Spinning Disk Unit&amp;nbsp; CSU-X1M1</td><td>Yokogawa</td><td></td><td></td></tr><tr><td>491 nm 50 mW laser</td><td>COBOLT CALYPSO</td><td></td><td></td></tr><tr><td>HCX PL APO CS Objectif</td><td>Leica</td><td></td><td>Objective lens ; Magnification 100X ; Numerical aperture 1.40 ; Immersion oil</td></tr><tr><td>CoolSnap HQ2 (FireWire) Camera</td><td>Photometrics</td><td></td><td>Pixel size 6.45 &amp;micro;m x 6.45 &amp;micro;m ; Definition 1,392 x 1,040 ; Encoding the image in 14 Bit</td></tr><tr><td>Metamorph 7.7.5 software</td><td>Molecular Devices</td><td></td><td>For the control of the microscope</td></tr><tr><td>GraphPad Prism software</td><td></td><td></td><td>For the statistics analysis</td></tr></tbody></table>

phagosome migration and velocity measured in live primary human macrophages infected with hiv-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human immunodeficiency virus (HIV)-1 and because of their resistance to its cytopathic effects they can be considered to be persistent viral reservoirs. In addition, HIV-infected macrophages exhibit defective functions that contribute to the development of opportunistic diseases.
The exact mechanism by which HIV-1 impairs the phagocytic response of macrophages was unknown. We had previously shown that the uptake of various particulate material by macrophages was inhibited when they were infected with HIV-1. This inhibition was only partial and phagosomes did form within HIV-infected macrophages. Therefore, we focused on analyzing the fate of these phagosomes. Phagosome maturation is accompanied by migration of these compartments towards the cell center, where they fuse with lysosomes, generating phagolysosomes, responsible for degradation of the ingested material. We used IgG-opsonized Sheep Red Blood Cells as a target for phagocytosis. To measure the speed of centripetal movement of phagosomes in individual HIV-infected macrophages, we used a combination of bright field and fluorescence confocal microscopy. We established a method to calculate the distance of phagosomes towards the nucleus, and then to calculate the velocity of the phagosomes. HIV-infected cells were identified thanks to a GFP-expressing virus, but the method is applicable to non-infected cells or any type of infection or treatment.

HIV-1感染および非感染hMDMsによる食作用をのFcR媒介性は、モデル標的としてのIgGオプソニン化SRBCを（ 図1）を使用してここで説明されています。このプロトコルの重要なステップは、hMDMsの準備とHIV-1の感染です。確かに、分化したマクロファージの収量と品質は、ドナーの間で変化するだけでなく、10から40%の範囲の効率で感染率。これは破片としての代...

Watch this Scientific Journal Video about Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1 at JoVE.com

Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes.

HIV-1に感染したライブ初代ヒトマクロファージで測定ファゴソームの移行と速度

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human ...

phagosome-migration-velocity-measured-live-primary-human-macrophages

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

8.1K ビュー.  Inserm U1016, Institut Cochin, CNRS UMR 8104, Université Paris Descartes, Sorbonne Paris Cité. We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes. 

ビデオ: Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

HIVとライブ共焦点顕微鏡の蛍光クローンを使用して、HIVの可視化細胞間伝達

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

免疫・感染学

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

真菌病原体の食作用の生細胞ビデオ顕微鏡

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

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 ...

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

マクロファージ食作用およびアクチン重合の評価のためのハイスループット蛍光技術

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

"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.

"Phagosome Closure Assay" 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 ...

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate hosts, respectively. While pathogenic symptoms of leishmaniasis can vary widely, from benign cutaneous lesions to highly fatal visceral disease forms depending on the infective species, all Leishmania species reside inside host macrophages during the vertebrate stage of their lifecycle. Leishmania infectivity is therefore directly related to its ability to invade, survive and replicate within parasitophorous vacuoles (PVs) inside macrophages. Thus, assessing the parasite&#39;s ability to replicate intracellularly serves as a dependable method for determining virulence. Studying leishmaniasis development using animal models is time-consuming, tedious and often difficult, particularly with the pathogenically important visceral forms. We describe here a methodology to follow the intracellular development of Leishmania in bone marrow-derived macrophages (BMMs). Intracellular parasite numbers are determined at 24 h intervals for 72 - 96 h following infection. This method allows for a reliable determination of the effects of various genetic factors on Leishmania virulence. As an example, we show how a single allele deletion of the Leishmania Mitochondrial Iron Transporter gene (LMIT1) impairs the ability of the Leishmania amazonensis mutant strain LMIT1/&#916;Lmit1 to grow inside BMMs, reflecting a drastic reduction in virulence compared to wild-type. This assay also allows precise control of experimental conditions, which can be individually manipulated to analyze the influence of various factors (nutrients, reactive oxygen species, etc.) on the host-pathogen interaction. Therefore, the appropriate execution and quantification of BMM infection studies provide a non-invasive, rapid, economical, safe and reliable alternative to conventional animal model studies.

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

All pathogenic Leishmania species reside and replicate inside macrophages of their vertebrate hosts. Here, we present a protocol to infect murine bone marrow-derived macrophages in culture with Leishmania, followed by precise quantification of intracellular growth kinetics. This method is useful for studying individual factors influencing host-pathogen interaction and Leishmania virulence.

細胞内マクロファージの内部成長の定量化は高速とリーシュ マニア原虫の病原性を評価するための信頼性の高い方法です。

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

マウスのマクロファージによる貪食のコマ撮りの 3 D イメージング

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up Leishmania parasites by phagocytosis, consisting of recognizing ligands on parasite surfaces by multiple host cell receptors. Binding of Leishmania to macrophage membranes occurs through complement receptor type 1 (CR1) and complement receptor type 3 (CR3) and Pattern Recognition Receptors. Lipophosphoglycan (LPG) and 63 kDa glycoprotein (gp63) are the main ligands involved in macrophage-Leishmania interactions. Following the initial recognition of parasite ligands by host cell receptors, parasites become internalized, survive, and multiply within parasitophorous vacuoles. The maturation process of Leishmania-induced vacuoles involves the acquisition of molecules from intracellular vesicles, including monomeric G protein Rab 5 and Rab 7, lysosomal associated membrane protein 1 (LAMP-1), lysosomal associated membrane protein 2 (LAMP-2), and microtubule-associated protein 1A/1B-light chain 3 (LC3).
Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells using confocal microscopy, including (i) binding (ii) internalization, and (iii) phagosome maturation. By adding to the body of knowledge surrounding these determinants of infection outcome, we hope to improve the understanding of the pathogenesis of Leishmania infection and support the eventual search for novel chemotherapeutic targets.

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

The mechanism associated with phagocytosis in Leishmania infection remains poorly understood. Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells.

共焦点顕微鏡を用いた リーシュマニア の貪食作用の調査

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up ...

Obtainment of Macrophages from Human Monocytes to Assess Leishmania braziliensis Infection Rate and Innate Host Immune Response

Macrophages are multifunctional cells essential to the immune system function, and the primary host cell in Leishmania braziliensis (Lb) infection. These cells are specialized in microorganism recognition and phagocytosis, but also activate other immune cells and present antigens, as well as promote inflammation and tissue repair. Here, we describe a protocol to obtain mononuclear cells from peripheral blood (PBMC) of healthy donors to separate monocytes that then differentiate into macrophages. These cells can then be infected in vitro at different Lb concentrations to evaluate the ability to control infection, as well as evaluate host cell immune response, which can be measured by several methods. PBMCs were first isolated by centrifuging with Ficoll-Hypaque gradient and then plated to allow monocytes to adhere to culture plates; non-adherent cells were removed by washing. Next, adherent cells were cultured with macrophage-colony stimulating factor (M-CSF) for 7 days to induce macrophage differentiation. We suggest plating 2 x 106&#160;cells per well on 24-well plates in order to obtain 2 x 105&#160;macrophages. Fully differentiated macrophages can then be infected with Lb for 4 or 24 hours. This protocol results in a significant percentage of infected cells, which can be assessed by optical or fluorescence microscopy. In addition to infection index, parasite load can be measured by counting the numbers of parasites inside each cell. Further molecular and functional assays can also be performed in culture supernatants or within the macrophages themselves, which allows this protocol to be applied in a variety of contexts and also adapted to other intracellular parasite species.

<meta charset="utf8"></head><body>Leishmania braziliensis感染率と自然宿主免疫応答を評価するためのヒト単球からのマクロファージの取得

This protocol describes the process for obtaining human macrophages from monocytes for infection with Leishmania braziliensis. It also allows researchers to evaluate infection rate and parasite viability, ROS production by fluorescence microscopy, and the production of inflammatory mediators in culture supernatants to investigate macrophage response to infection.

Leishmania braziliensis感染率と自然宿主免疫応答を評価するためのヒト単球からのマクロファージの取得

Macrophages are multifunctional cells essential to the immune system function, and the primary host cell in Leishmania braziliensis (Lb) infection. These ...

HIV-1に感染したライブ初代ヒトマクロファージで測定ファゴソームの移行と速度

要約

さらに動画を探す

HIVとライブ共焦点顕微鏡の蛍光クローンを使用して、HIVの可視化細胞間伝達

真菌病原体の食作用の生細胞ビデオ顕微鏡

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

マクロファージ食作用およびアクチン重合の評価のためのハイスループット蛍光技術

「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

細胞内マクロファージの内部成長の定量化は高速とリーシュマニア原虫の病原性を評価するための信頼性の高い方法です。

マウスのマクロファージによる貪食のコマ撮りの 3 D イメージング

共焦点顕微鏡を用いたリーシュマニアの貪食作用の調査

Leishmania braziliensis感染率と自然宿主免疫応答を評価するためのヒト単球からのマクロファージの取得

Leishmania braziliensis感染率と自然宿主免疫応答を評価するためのヒト単球からのマクロファージの取得

HIV-1に感染したライブ初代ヒトマクロファージで測定ファゴソームの移行と速度

要約

さらに動画を探す

HIVとライブ共焦点顕微鏡の蛍光クローンを使用して、HIVの可視化細胞間伝達

真菌病原体の食作用の生細胞ビデオ顕微鏡

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

マクロファージ食作用およびアクチン重合の評価のためのハイスループット蛍光技術

「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

細胞内マクロファージの内部成長の定量化は高速とリーシュ マニア原虫の病原性を評価するための信頼性の高い方法です。

マウスのマクロファージによる貪食のコマ撮りの 3 D イメージング

共焦点顕微鏡を用いた リーシュマニア の貪食作用の調査

Leishmania braziliensis感染率と自然宿主免疫応答を評価するためのヒト単球からのマクロファージの取得

Leishmania braziliensis感染率と自然宿主免疫応答を評価するためのヒト単球からのマクロファージの取得

細胞内マクロファージの内部成長の定量化は高速とリーシュマニア原虫の病原性を評価するための信頼性の高い方法です。

共焦点顕微鏡を用いたリーシュマニアの貪食作用の調査