This work was funded by both the&#160;Canadian Institutes of Health Research&#160;and&#160;Natural Sciences and the Engineering Research Council of Canada. M. Bisaillon is a Chercheur Boursier Senior from the Fonds de Recherche en Sant&#233; du Qu&#233;bec and also a member of the Centre de Recherche Clinique du Centre Hospitalier Universitaire de Sherbrooke. We thank Dr. Ralf Bartenschlager for the generous gift of the HCV replicon system. We also thank Dr. Charles Rice and Dr. Daniel Lamarre for kindly providing the hepatic cell line. We also want to thank Guillaume Tremblay for technical assistance.

1.細胞の調製および治療<ol><li> 24ウェルプレートにおいて、ウェルの底部にカバーガラスを置きます。リン酸緩衝生理食塩水（PBS）を各ウェルを洗浄します。 </li><li> /ウェル5×10 4細胞の密度に各ウェル中のHCVサブゲノムレプリコンを支援するシードのHuh-7細胞。行うには治療法がない場合、細胞は、より高い密度で播種することができます。培地は、ダルベッコ改変イーグル培地、10％ウシ胎児血清（FBS）、2mMのL-グルタミン、1mMピルビン酸ナトリウムおよび250 mg / mlのG-418（レプリコンを維持するため）を補足した（DMEM）です。 </li><li> 37℃で培養し、5％CO 2翌日、ヒトの乳から精製されたHLFの3μMで細胞を処理します。 </li></ol> 2.パラホルムアルデヒド/ショ糖の製造（12％PAFおよび12％ショ糖） <ol><li>ビーカーに500mlのPBSに入れ、20°Cと30°Cの間に加熱します。 PBS中のショ糖の60グラムを溶解します。 paraformalの60グラムを溶かしPBS /スクロース溶液中のアルデヒド（PAF）。透明な溶液が得られるまでゆっくりと2NのNaOH（3~7 ml）を加えます。 </li><li> HClでpHを7.4に調整します。 PBSで500ミリリットルに、完全なボリューム。ワットマンのフィルター上の重力によってフィルタします。使用前に、4％PAFおよび4％スクロース（ストレージ1週間に最大4°C）の最終濃度にPBSで溶液を希釈します。 </li></ol> 3.免疫蛍光<ol><li>所望の時間（0時間、2時間または治療の24時間）で、PBSで2回（1分）細胞を洗浄し、室温で20分間、PBS / 4％PAF / 4％スクロースで固定します。細胞が30-40％コンフルエントであり、典型的には1.5×10 5個の細胞が使用されます。 </li><li> 20分間、PBS中10％正常ヤギ血清（NGS）中で室温で5分間、PBS中の0.15％のトリトンX-100で細胞を透過性とブロック。 </li><li> 10％NGS中で目的のタンパク質の一次抗体を希釈する（例：1,000：一次マウス抗HLF抗体の1希釈）。細胞に一次抗体を適用します。 </li><li> RTまたはOで4時間後/4℃でのNは、10％NGSで5分間、細胞を3回洗浄します。 10％NGS中で希釈した蛍光色素で標識した二次抗体を用いた細胞染色（例：蛍光色素を抗体抗マウス1に連結された488nmでの励起波長で1,000）暗所でRTで1時間。 </li><li> PBSで5分間、細胞を1回洗浄し、暗所で室温で15分間、4 &#39;、6-ジアミジノ-2-フェニルインドール（DAPI）（1μg/ ml）で細胞を染色します。 </li><li> PBSで5分間、細胞を2回洗浄します。マウントは、顕微鏡を通して視覚化する前にメディアをマウントSlowFadeのメガネをカバーしています。細胞は今落射蛍光または共焦点顕微鏡による分析のために準備ができています。 </li><li>検出の特異性を確保するために、別のコントロールを使用します。例えば、すべての抗体は、自己蛍光を検出するために省略することができます。一次抗体を、二次抗体による非特異的染色を決定するために省略することができます。ない最後に、可能な場合は、コントロール細胞または組織目的の分子を用いることも可能で発現します。 </li><li>使用される蛍光標識に適した適切な蛍光顕微鏡（落射蛍光または共焦点）とフィルターセットを使用してサンプルを可視化します。スライドはまた、&lt;-20°Cの場合、後の検査でスライドボックスに保存することができます。 </li></ol> 4.蛍光顕微鏡<ol><li>光退色を防止するために、暗闇の中でサンプルを保管してください。選択した顕微鏡（落射蛍光または共焦点）の光源をオンにします。 </li><li>顕微鏡の電源をオンにします。顕微鏡用カメラの電源をオンにします。可視化のための顕微鏡のスライドを入れてください。対物レンズの開口数を増加させるために、スライド上の液浸油を加えます。 </li><li>適切なフィルタを選択します。フォーカスし、適切な目標を調整します。適切な蛍光色素を検出するが光漂白を防ぐためにシャッターを調整します。サンプルを見ているときに、背景光を最小化するためにオーバーヘッドライトをオフにしてください。 </li><li>目を変更電子フィルタは、（例えばDAPIおよび488nmでの励起波長のための）様々な蛍光色素を可視化します。カメラで写真を撮ります。 </li></ol>

<ol>
	<li>Odell, I. D., Cook, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+techniques.">Immunofluorescence techniques.</a> J Invest Derm. 133 (1), e4 (2013).</li><li>Portugal, J., Waring, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assignment+of+DNA+binding+sites+for+4&#8217;,6-diamidine-2-phenylindole+and+bisbenzimide+(Hoechst+33258).+A+comparative+footprinting+study.">Assignment of DNA binding sites for 4&#8217;,6-diamidine-2-phenylindole and bisbenzimide (Hoechst 33258). A comparative footprinting study.</a> Biochim Biophys Acta. 949 (2), 158-168 (1988).</li><li>Lichtman, J. W., Conchello, J. -. A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+microscopy.">Fluorescence microscopy.</a> Nat Methods. 2 (12), 910-919 (2005).</li><li>St Croix, C. M., Shand, S. H., Watkins, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+microscopy:+comparisons,+applications,+and+problems.">Confocal microscopy: comparisons, applications, and problems.</a> BioTechniques. 39, S2-S5 (2005).</li><li>Yi, M., Kaneko, S., Yu, D. Y., Murakami, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+C+virus+envelope+proteins+bind+lactoferrin.">Hepatitis C virus envelope proteins bind lactoferrin.</a> Journal of virology. 71, 5997-6002 (1997).</li><li>Wakabayashi, H., Oda, H., Yamauchi, K., Abe, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lactoferrin+for+prevention+of+common+viral+infections.">Lactoferrin for prevention of common viral infections.</a> J Inf Chem. 20 (11), 666-671 (2014).</li><li>Picard-Jean, F., Bouchard, S., Lariv&#233;e, G., Bisaillon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intracellular+inhibition+of+HCV+replication+represents+a+novel+mechanism+of+action+by+the+innate+immune+Lactoferrin+protein.">The intracellular inhibition of HCV replication represents a novel mechanism of action by the innate immune Lactoferrin protein.</a> Antiviral research. 111, 13-22 (2014).</li><li>Johnson, G. D., Davidson, R. S., McNamee, K. C., Russell, G., Goodwin, D., Holborow, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fading+of+immunofluorescence+during+microscopy:+a+study+of+the+phenomenon+and+its+remedy.">Fading of immunofluorescence during microscopy: a study of the phenomenon and its remedy.</a> J Immunol Methods. 55 (2), 231-242 (1982).</li><li>Green Remington, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=fluorescent+protein:+a+perspective.">fluorescent protein: a perspective.</a> Protein Sci. 20 (9), 1509-1519 (2011).</li></ol>

The authors declare that they have no competing financial interests.

HCVの流行は、肝硬変、肝不全や肝細胞癌の危険にそれらを置くこと、新たに感染した患者の80％が慢性感染症を発症して、世界的な脅威のまま。最近2 NS3 N末端プロテアーゼ阻害剤（ボセプレビルおよびテラプレビル）の規制当局の承認によって実証されるようにHCV複製および成熟をターゲットに直接作用型抗ウイルス剤は、プライム抗HCV剤を表します。 HLF抗HCV活性は、現在、ビリオンを循環し、その肝細胞標的に侵入するのを防止する結合、ウイルス侵入阻害剤として挙動すると考えられています。 HLFの我々の研究は、（細胞外ビリオンの中和は異なる）新規の作用、細胞内の抗HCV 機構 7 を提示します。 免疫蛍光を効率的に生物学的サンプル中の標的分子の分布および/または局在化を可視化するために使用することができます。他の多くの蛍光技術、主要なプロの1に見られるように免疫蛍光法に関連したblemsは、（蛍光色素の光化学破壊）8を光退色されます。光退色に起因する活性の損失は、光退色または強度または露光の時間スパンを減らすことによってより少ない傾向があり、より堅牢な蛍光色素を使用することによってのいずれかで回避することができます。抗体は、細胞膜を横切ることができないので、免疫蛍光法は、一般的に固定された細胞にのみ限定されます。生細胞におけるタンパク質の分布/局在化を監視するための別のアプローチは、従って、一般に、緑色蛍光タンパク質（GFP）9と蛍光タンパク質ドメインを含むタンパク質の発現に依存しています。しかし、このようなGFPエピトープの付加はしばしば、タンパク質の局在化及び活性の改変に関連しています。免疫蛍光は、それがまた頻繁に監視するために使用される組織切片、培養細胞株、または個々の細胞を含む種々のサンプル数にも使用することができタンパク質、グリカン、および小分子の局在/分布。この技術の主な利点は、その単純さと、それが蛍光染色（例えば、DNA染色）の他、非抗体法と組み合わせて使用することができるという事実に存在します。また、商業的に製造した二次抗体は、色の広大な配列で、比較的安価で入手できます。 現在の研究では、HCVサブゲノムレプリコンをサポートする肝細胞の免疫蛍光は、HLFで処理して、HCVの染色強度の質的低下を明らかにしました。このサブゲノムレプリコンシステムは、HLFの一般に受け入れられている薬理学的標的であり、HCV E1およびE2エンベロープ糖タンパク質を、欠けているように、これは興味深い観察です。細胞外のビリオンの中和活性とは異なり、HLFは、ウイルス複製を損なうために、HCVに感染した肝細胞によって内在化される必要があります。 fuorescence顕微鏡の使用は、私たちはevaluを許可この可能性を食べました。 HCVに感染した肝細胞のプレインキュベーションの際に、外因性HLFが正常に内在化し、細胞内のウイルスの複製を損なうことが示されました。この小説HLF抗ウイルス活性を理解することは、この自然免疫タンパク質の阻害のグローバル（および多面的な可能性が高い）のメカニズムの理解に一歩前進を構成しています。

Immunofluorescence is a technique that uses a fluorescence microscope to visualize the distribution and/or localization of a target molecule in a biological sample. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell1. It is typically used on tissue sections and cultured cell lines in order to analyze the distribution/localization of various biological molecules such as proteins, nucleic acids and glycans. It should be noted that immunofluorescence is often used in combination with other non-antibody methods of fluorescent staining such as the 4',6-diamidino-2-phenylindole (DAPI) stains which are typically used to label DNA2. Moreover, this technique involves fixation of the cells which allows the analysis of cells at a specific time.
Different types of microscopes can be used to analyze immunofluorescence samples. The simplest is the epifluorescence microscope (Figure 1) for which excitation of the fluorochrome and detection of the fluorescence are done through the same light path3. Because most of the excitation light is transmitted through the sample, only reflected excitatory light can reach the objective together with the emitted light. This approach unfortunately leadsto a frequent high signal to noise ratio.In contrast, confocal microscopy (Figure 2) offers a distinct advantage for increasing optical resolution and contrast by means of adding a spatial pinhole placed at the confocal plane of the lens to eliminate out-of-focus light4. This approach allows the reconstruction of three-dimensional structures from the obtained images. However, since an important fraction of the light from the sample is blocked at the pinhole, long exposures are often required.
There are two classes of immunofluorescence techniques, primary (or direct) and secondary (or indirect). Direct immunofluorescence involves a primary antibody linked with a fluorochrome (Figure 3). This method is less frequently used because it provides lower signal, involves higher cost and less flexibility1. Moreover, such antibodies are generally harder to find commercially. On the other hand, the direct attachment of the fluorochrome to the antibody significantly reduces the number of steps in the procedure, saving time and frequently reducing non-specific background signal. This also limits the possibility of antibody cross-reactivty.
Indirect immunofluorescence involves a primary unlabelled antibody which is specific for the epitope of interest1. A secondary antibody which carries the fluorochrome then recognizes the primary antibody and binds to it (Figure 3). Although indirect immunofluorescence is more complex and time consuming than direct immunofluorescence, it is frequently used because of its high sensitivity and it also provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In addition, a vast array of commercial secondary antibodies is available at affordable prices.
Hepatitis C virus (HCV) is a major public-health problem with 130-170 million individuals chronically infected worldwide. In order to halt the epidemic, therapy against HCV will need to be both effective and widely available. Studies focusing on safe and affordable natural product active against HCV have revealed the antiviral activity of the human Lactoferrin (hLF) protein which binds and neutralizes the circulating virion5. In the current study, investigation of hLF activity on the HCV subgenomic replicon system, which is independent from viral entry and shedding, revealed a distinct antireplicative activity of hLF against HCV. This manuscript presents a study in which immunofluorescence assays were performed to monitor the internalization of hLF, an important component of the immune system6, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus (HCV).

<table><tbody><tr><td>DMEM</td><td>Wisent</td><td>319-005-CL</td><td></td></tr><tr><td>PAF</td><td>BioShop</td><td>PAR070.1</td><td>Flammable solid, skin irritant, lungs and eyes&amp;nbsp;</td></tr><tr><td>PBS</td><td>Wisent</td><td>311-425-CL</td><td>Without Ca&lt;sup&gt;2+ &lt;/sup&gt;&amp;amp; Mg&lt;sup&gt;2+&lt;/sup&gt;</td></tr><tr><td>NGS</td><td>Wisent</td><td>053-150</td><td></td></tr><tr><td>AlexaFluor 488-labeled anti-mouse</td><td>Invitrogen</td><td>A11017</td><td></td></tr><tr><td>AlexaFluor 568-labeled anti-rabbit</td><td>Invitrogeb</td><td>A21069</td><td></td></tr><tr><td>Wheat germ agglutinin Alexa Fluor 488 conjugate (WGA)</td><td>Invitrogen</td><td>W11261</td><td>Potentially mutagenic</td></tr><tr><td>Anti-NS5A rabbit</td><td>Abcam</td><td>ab2594</td><td></td></tr><tr><td>Anti-hLF mouse</td><td>Abcam</td><td>ab10110</td><td></td></tr><tr><td>SlowFade</td><td>Invitrogen</td><td>S36937</td><td></td></tr><tr><td>Hoechst stain</td><td>Life Techn.</td><td>H1399</td><td>Potentially&amp;nbsp;mutagenic&amp;nbsp;
and carcinogenic</td></tr><tr><td>hLF</td><td>Sigma</td><td>L0520</td><td></td></tr><tr><td>Nikon Eclipse visible/epifluorescence Microscope</td><td>Nikon</td><td>TE2000-E</td><td></td></tr><tr><td>epifluorescence/confocal microscope</td><td>Olympus</td><td>FV1000</td><td></td></tr></tbody></table>

immunofluorescence to monitor the cellular uptake of human lactoferrin and its associated antiviral activity against the hepatitis c virus

免疫蛍光は、一般的に生物学の多くの側面を研究するために使用される実験技術です。それは、典型的には、細胞及び組織中の標的分子の分布および/または局在化を可視化するために使用されます。免疫蛍光は、細胞内でのそれらの対応する抗原に対する蛍光標識抗体の特異性に依存しています。直接的および間接的の両方の免疫アプローチは、蛍光色素に結合された抗体の使用に依存しているものを用いることができます。それは、より低い信号を提供し、より高いコスト、より少ない柔軟性を必要とするので、直接免疫蛍光法は、あまり頻繁に使用されます。対照的に、間接免疫蛍光法は、より一般的に、その高い感度を使用し、複数の二次抗体は、それぞれ一次抗体に付着することができるので、増幅された信号を供給する。本稿では、落射蛍光顕微鏡及び共焦点顕微鏡法の双方は、ヒトラクトフェリンの内在化、免疫の重要な構成要素を監視するために使用しました。肝細胞へのシステム。また、我々は、免疫蛍光法を用いて、C型肝炎ウイルスの細胞内複製にHLFの阻害可能性を監視しました。これらのアプローチに関連する利点と欠点の両方が議論されています。

外因的に投与さHLFを内在する肝臓のHuh-7細胞株の能力を、共焦点miscroscopeに免疫蛍光を用いてモニターしました。 HLFは、培養培地に添加し、インターナリゼーションは、その後、細胞外HLFをPBSで洗浄し、結合したHLF残留膜を5分間トリプシン処理（1 ml）で分解し、24時間おきました。細胞は、再播種し、免疫蛍光染色の前に18時間再接着させました。細胞質の限界を概説するために、細胞膜を488nmの複合体の励起波長のコムギ胚芽凝集素（WGA）蛍光色素で染色しました。 HLF染色は抗HLF一次抗体と二次抗体に結合された波長633nmの蛍光色素を使用して達成されました。 HLFの正確な位置を決定するために、細胞を、共焦点顕微鏡によるシーケンシャル0.5μmの水平断面次画像化した。 図4は、オプトインを示す図ミッドセル厚に対応するicalの断面。外因性HLFの添加後、HLFのかなりの量は、肝細胞の細胞質中に検出されました。同一の楽器の設定を使用しない時HLFは、未処理の細胞の内部観察されませんでした。一緒に、これらのデータは、肝細胞が、それらの細胞外環境からHLFを取得する能力を持っていることを確認します。 HCVの細胞内複製を阻害するHLFの能力は、次のエピ蛍光顕微鏡を用いてモニターしました。複製（HCV NS3、NS4A、NS4B、NS5A、NS5Bタンパク質を発現する）HCVサブゲノムレプリコンを支持するHuh-7細胞を、0,2または24時間、3μMHLFで処理しました。 HLF及びHCV NS5Aタンパク質についての二重免疫蛍光染色は、落射蛍光顕微鏡法により分析しました。 HLFで処理した細胞において減少した（HCV非構造（a）タンパク質の免疫蛍光によって測定される）これらの結果は、HCVサブゲノムレプリコンの染色を示す。 図5 </s<html> trong&gt; HLFの処理の24時間後のHCV染色強度の約50％の質的低下を明らかにしています。このサブゲノムレプリコンシステムは、HLFの一般に受け入れられている薬理学的標的であり、HCV E1およびE2エンベロープ糖タンパク質を、欠けているように、これは興味深い観察です。 HCV染色の退色との併用は、HLFの蓄積は、2時間後に検出されるようになった、と強いHLFの蓄積は24時間後に観察されました。 <img alt="図1" src="/files/ftp_upload/53053/53053fig1.jpg" /> 落射蛍光顕微鏡の原理図1。顕微鏡のこのタイプでは、励起フィルターは（蛍光標識抗体を保有する織物博物館や細胞）試料を励起する特定波長を選択します。また、発光フィルターは、試料を標識するために使用されるフ​​ルオロフォアの発光スペクトル特性と一致するように選択されます。ロード/ 53053 / 53053fig1large.jpg「ターゲット= &quot;_空白&quot;&gt;この図の拡大版をご覧になるにはこちらをクリックしてください。 <img alt="図2" src="/files/ftp_upload/53053/53053fig2.jpg" /> 共焦点顕微鏡の原理図2。顕微鏡のこのタイプは、焦点外の光信号を除去するために、発光ピンホールを使用しています。焦点面に非常に近い蛍光によって生成された光のみを検出することができるので、画像の光学解像度は、落射蛍光顕微鏡のそれよりもはるかに優れています。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53053/53053fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図3" src="/files/ftp_upload/53053/53053fig3.jpg" /> 間接および直接免疫蛍光の図3の回路図。直接免疫蛍光法が含まれます目的の分子（一次抗体）に特異的である蛍光色素で標識された抗体の一種類のみ。これとは対照的に、間接免疫蛍光法は、非標識一次抗体の種に固有の蛍光色素で標識した二次抗体を必要とします。複数の二次抗体は、蛍光シグナルを増加させ、各一次抗体に付加することができます。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53053/53053fig3large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図4" src="/files/ftp_upload/53053/53053fig4.jpg" /> 外因性HLFの図4.肝細胞の取り込み。ホ-7細胞を、24時間、0μMまたは3μMのHLFで処理された二回洗浄し、5分間のトリプシン処理に提出し、18時間のために再付着させ、免疫蛍光法に供しました。核はワット染色しました、プラズマ膜は、WGA（グリーンチャンネル）とHLFで標識したi番目のDAPI（青チャネル）は、抗HLF抗体（赤チャンネル）で染色しました。画像は、60倍の元の倍率で共焦点顕微鏡により得ました。水平光学断面は、セルの中央の厚さを表します。スケールバーは10μm。 （11）から変更された。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53053/53053fig4large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="Figure5" src="/files/ftp_upload/53053/53053fig5.jpg" /> 図5 HLF処置はHCV染色を減少させる。HCVサブゲノムレプリコンの複製を支持するHuh-7細胞を、0,2、または24時間、3μMHLFで処理し、免疫蛍光に供しました。核はDAPI（青チャネル）で染色した、HCV抗NS5A抗体（赤チャンネル）によって明らかにされたとHLFは、抗HLF Aで染色しましたntibody（グリーンチャンネル）。画像は60倍の倍率で、元の落射蛍光顕微鏡で取得しました。スケールバーは10μm。 （11）から変更された。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53053/53053fig5large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus at JoVE.com

Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.

C型肝炎ウイルスに対するヒトラクトフェリンの細胞取り込みおよびそれに関連する抗ウイルス活性を監視するために、免疫蛍光

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or ...

immunofluorescence-to-monitor-cellular-uptake-human-lactoferrin-its

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

13.3K ビュー.  Université de Sherbrooke. The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.

ビデオ: Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

インフルエンザウイルスのためのハイスループット検出方法

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

免疫・感染学

Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in human non-hepatic and murine cell lines, the efficiency was very low and required long-term selection procedures using HCV replicon constructs expressing dominant antibiotic-selectable markers1-5. HCV in vitro research is therefore limited to human hepatoma cell lines permissive for virus entry and completion of the viral life cycle. Due to HCVs narrow species tropism, there is no immunocompetent small animal model available that sustains the complete HCV replication cycle 6-8. Inefficient replication of HCV in non-human cells e.g. of mouse origin is likely due to lack of genetic incompatibility of essential host dependency factors and/or expression of restriction factors.
We investigated whether HCV propagation is suppressed by dominant restriction factors in either human cell lines derived from non-hepatic tissues or in mouse liver cell lines. To this end, we developed two independent conditional trans-complementation methods relying on somatic cell fusion. In both cases, completion of the viral replication cycle is only possible in the heterokaryons. Consequently, successful trans-complementation, which is determined by measuring de novo production of infectious viral progeny, indicates absence of dominant restrictions.
Specifically, subgenomic HCV replicons carrying a luciferase transgene were transfected into highly permissive human hepatoma cells (Huh-7.5 cells). Subsequently, these cells were co-cultured and fused to various human and murine cells expressing HCV structural proteins core, envelope 1 and 2 (E1, E2) and accessory proteins p7 and NS2. Provided that cell fusion was initiated by treatment with polyethylene-glycol (PEG), the culture released infectious viral particles which infected na&iuml;ve cells in a receptor-dependent fashion.
To assess the influence of dominant restrictions on the complete viral life cycle including cell entry, RNA translation, replication and virus assembly, we took advantage of a human liver cell line (Huh-7 Lunet N cells 9) which lacks endogenous expression of CD81, an essential entry factor of HCV. In the absence of ectopically expressed CD81, these cells are essentially refractory to HCV infection 10 . Importantly, when co-cultured and fused with cells that express human CD81 but lack at least another crucial cell entry factor (i.e. SR-BI, CLDN1, OCLN), only the resulting heterokaryons display the complete set of HCV entry factors requisite for infection. Therefore, to analyze if dominant restriction factors suppress completion of the HCV replication cycle, we fused Lunet N cells with various cells from human and mouse origin which fulfill the above mentioned criteria. When co-cultured cells were transfected with a highly fusogenic viral envelope protein mutant of the prototype foamy virus (PFV11) and subsequently challenged with infectious HCV particles (HCVcc), de novo production of infectious virus was observed. This indicates that HCV successfully completed its replication cycle in heterokaryons thus ruling out expression of dominant restriction factors in these cell lines. These novel conditional trans-complementation methods will be useful to screen a large panel of cell lines and primary cells for expression of HCV-specific dominant restriction factors.

We describe two methods for conditional trans-complementation of hepatitis C virus (HCV) assembly and the completion of the full viral life cycle, which rely on heterokaryon formation. These techniques are suitable to screen for cell lines that express dominant restriction factors, which preclude production of infectious HCV progeny.

HCVの制限要因を発見するヘテロカリオンの形成の2つの方法

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in ...

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

C型肝炎ウイルスの複製を分析するためのプロトコル

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

インフルエンザ血球凝集素抗体による Fc を介したエフェクター機能を評価する方法

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

蛍光によるウイルスRNAの検出<em&gt;その場で</em&gt;ハイブリダイゼーション（FISH）

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

生物学

A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, studying IAV requires the use of laborious secondary approaches to detect the presence of the virus in infected cells and/or in animal models of infection. This limitation has been recently circumvented with the generation of recombinant IAVs expressing easily traceable fluorescent or bioluminescent (luciferase) reporter proteins. However, researchers have been forced to select fluorescent or luciferase reporter genes due to the restricted capacity of the IAV genome for including foreign sequences. To overcome this limitation, we have generated a recombinant replication-competent bi-reporter IAV (BIRFLU) stably expressing both a fluorescent and a luciferase reporter gene to easily track IAV infections in vitro and in vivo. To this end, the viral non-structural (NS) and hemagglutinin (HA) viral segments of influenza A/Puerto Rico/8/34 H1N1 (PR8) were modified to encode the fluorescent Venus and the bioluminescent Nanoluc luciferase proteins, respectively. Here, we describe the use of BIRFLU in a mouse model of IAV infection and the detection of both reporter genes using an in vivo imaging system. Notably, we have observed a good correlation between the expressions of both reporters and viral replication. The combination of cutting-edge techniques in molecular biology, animal research and imaging technologies, provides researchers the unique opportunity to use this tool for influenza research, including the study of virus-host interactions and dynamics of viral infections. Importantly, the feasibility to genetically alter the viral genome to express two foreign genes from different viral segments opens up opportunities to use this approach for: (i) the development of novel IAV vaccines, (ii) the generation of recombinant IAVs that can be used as vaccine vectors for the treatment of other human pathogen infections.

Influenza A viruses (IAVs) are contagious respiratory pathogens that cause annual epidemics and occasional pandemics. Here, we describe a protocol to track viral infections in vivo using a novel recombinant luciferase and fluorescence-expressing bi-reporter IAV (BIRFLU). This approach provides researchers with an excellent tool to study IAV in vivo.

ウイルス感染のライブイメージングと定量化のためのルシフェラーゼ蛍光レポーターインフルエンザウイルス

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, ...

Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that enable sensitive and simultaneous labeling of viral nucleic acid and protein. Conventional DNA fluorescence in situ hybridization (FISH) methods are often not compatible with immunostaining. We have therefore developed an imaging approach, MICDDRP (multiplex immunofluorescent cell-based detection of DNA, RNA and protein), which enables simultaneous single-cell visualization of DNA, RNA, and protein. Compared to conventional DNA FISH, MICDDRP utilizes branched DNA (bDNA) ISH technology, which dramatically improves oligonucleotide probe sensitivity and detection. Small modifications of MICDDRP enable imaging of viral proteins concomitantly with nucleic acids (RNA or DNA) of different strandedness. We have applied these protocols to study the life cycles of multiple viral pathogens, including human immunodeficiency virus (HIV)-1, human T-lymphotropic virus (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZKV), and influenza A virus (IAV). We demonstrated that we can efficiently label viral nucleic acids and proteins across a diverse range of viruses. These studies can provide us with improved mechanistic understanding of multiple viral systems, and in addition, serve as a template for application of multiplexed fluorescence imaging of DNA, RNA, and protein across a broad spectrum of cellular systems.

Presented here is a protocol for a fluorescence imaging approach, multiplex immunofluorescent cell-based detection of DNA, RNA, and protein (MICDDRP), a method capable of simultaneous fluorescence single-cell visualization of viral protein and nucleic acids of different type and strandedness. This approach can be applied to a diverse range of systems.

ウイルスの核酸とタンパク質を視覚化し、HIV、HTLV、HBV、HCV、ジカウイルス、インフルエンザ感染を監視するためのシングルセルマルチプレックス蛍光イメージング

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that ...

Measuring the Activation of Fc-Mediated Effector Functions by HA Antibodies

Source: Bailey, M. J., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56256">A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies.</a> J. Vis. Exp. (2018).
This video demonstrates an assay measuring the antibody-mediated activation of T cell effector functions. Adding an influenza virus hemagglutinin (HA)-specific monoclonal antibody to transfected mammalian cells expressing HA results in the formation of immune complexes with HA. Upon introducing engineered T cells expressing Fc receptors and a luciferase reporter, the T cells are activated by the antibody-HA complexes, and this activation is detected by adding a luciferase substrate and measuring the produced bioluminescence.

HA抗体によるFc媒介エフェクター機能の活性化の測定

1. Expression of Influenza Virus Hemagglutinin via Transfection

	Plate Human Embryonic Kidney (HEK 293T) cells at a density of 2 x 104 cells/well in a ...

JoVE Encyclopedia of Experiments

免疫学

抗体ベースの技術

特性評価と機能分析

An Immunofluorescence Method to Measure the Neutralization of Human Cytomegalovirus

This video demonstrates an immunofluorescence assay designed to measure the neutralization of Human Cytomegalovirus (HCMV). Virus-specific antibodies are mixed with the virus and incubated with human fibroblast cells. The antibodies neutralize the virus, rendering it unable to infect the host cells, while non-neutralized viruses infect the cells. After incubation, the extent of neutralization is determined by quantifying the virus-infected cells using immunofluorescence.

ヒトサイトメガロウイルスの中和を測定するための免疫蛍光法

1. Preparation of target cells for infection

	Seed target cells the day prior to performing the experiment in a 96-well plate (the &#39;target&#39; ...

免疫診断

画像化および視覚化技術

A Viral Attachment Assay for Screening of Antiviral Compounds

Source: Tai, C. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/53124">Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds.</a> J. Vis. Exp.&#160;(2015)
This video demonstrates a viral attachment assay for screening antiviral compounds. Using genetically modified hepatitis C virus and luciferase, it assesses inhibitory effects on viral attachment, as indicated by lower light intensity in co-treated wells compared to wells treated with the virus alone.

抗ウイルス化合物のスクリーニングのためのウイルス付着アッセイ

NOTE: Ensure that all procedures involving cell culture and virus infection are conducted in certified biosafety hoods that are appropriate for the ...