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. 세포 준비 및 치료 <ol><li> 24 웰 플레이트에서 웰의 바닥에 유리 덮개를 넣고. 인산염 완충 식염수 (PBS)를 각 웰을 세척 할 것. </li><li> / 웰의 5 × 104 세포의 농도로 각 웰에 subgenomic HCV 레 플리 콘을지지 시드 응-7 세포. 그럴 치료가 없으면 세포는 고밀도로 접종 될 수있다. 배양 배지는 둘 베코 변형 이글 중간 10 % 우태 혈청 (FBS)를 2mM L- 글루타민, 1 mM 피루브산 나트륨 및 250 ㎎ / ㎖ G-418 (레 플리 콘을 유지하기 위해)와 보충 (DMEM)이다. </li><li> 37 ° C에서 품어 5 % CO 2. 다음날, 인간의 우유에서 정제 HLF의 3 μm의와 세포를 치료. </li></ol> 2. 파라 포름 알데히드 / 자당 준비 (12 % PAF 12 % 자당) <ol><li> 20 ° C와 30 ° C 사이를 비커에 PBS 500 ㎖에 넣고 가열한다. PBS에서 자당의 60g을 녹인다. paraformal의 60g을 녹여PBS / 자당 용액에 알데히드 (PAF). 서서히 투명 용액이 얻어 질 때까지 2 N NaOH로 (3 내지 7 ml)에 추가한다. </li><li> HCl을 7.4으로 산도를 조정합니다. PBS로 500 ml의 완전한 볼륨. 워트 먼지 필터에 중력에 의해 필터링합니다. 사용 전에, PAF 4 % 4 % 자당 (스토리지 일주 최대 4 ℃)의 최종 농도로 PBS 용액으로 희석. </li></ol> 3. 면역 형광 <ol><li> 원하는 시간에 (0 시간은 2 시간 24 처리의 시간), PBS로 (1 분) 두 번 세포를 씻어 실온에서 20 분 동안 PBS / 4 % PAF / 4 % 자당에 고정합니다. 세포는 30 % ~ 40 % 합류에 통상적이며 1.5 × 105 세포가 사용된다. </li><li> 20 분 동안 PBS 중 10 % 정상 염소 혈청 (NGS)에서 RT에서 5 분 동안 PBS에 0.​​15 %의 트리톤 X-100 세포 및 블록 Permeabilize 하시려면. </li><li> 10 % NGS의 관심 단백질의 일차 항체를 희석 (예 : 1000 : 일차 마우스 항 HLF 항체 희석은 1). 세포에 차 항체를 적용합니다. </li><li> RT 또는 O에서 4 시간 후 /4 ° C에서 N은 10 % NGS 5 분 동안 세포를 세 번 씻는다. 10 % NGS 희석 형광 색소로 표지 된 이차 항체와 세포 얼룩 (예 : 형광 색소를 항 - 마우스 항체 (1)에 연결된 488 nm 인 여기 파장 : 1,000) 어두운 RT에서 1 시간 동안. </li><li> PBS로 5 분 동안 한 번 세포를 씻으과 어둠 속에서 실온에서 15 분 동안 4 &#39;, 6 diamidino -2- 페닐 인돌 (DAPI) (1 μg의 / ml)로 세포를 얼룩. </li><li> PBS로 5 분 동안 두 번 세포를 씻으십시오. 마운트 현미경을 통해 시각화하기 전에 SlowFade 설치 매체에 안경을 커버합니다. 셀은 표면 형광 또는 공 초점 현미경에 의한 분석을위한 준비가되어 있습니다. </li><li> 검출의 특이성을 보장하기 위해 다른 컨트롤을 사용합니다. 예를 들어, 모든 항체는자가 형광을 검출하기 위해 생략 될 수있다. 일차 항체는 이차 항체에 의한 비특이적 염색을 결정을 생략 할 수있다. 가능하면 마지막으로, 그렇지 않은 세포 또는 조직을 제어대상 분자가 또한 사용될 수 표현한다. </li><li> 사용되는 형광 표지에 적합한 적절한 형광 현미경 (또는 표면 형광 공 초점) 및 필터 세트를 이용하여 샘​​플을 시각화. 슬라이드는 또한 &lt;-20 ° C에 대한 시험에서 나중에 슬라이드 박스에 저장 될 수있다. </li></ol> 4. 형광 현미경 <ol><li> 광표백을 방지하기 위해 어둠 속에서 샘플을 보관하십시오. 선택한 현미경 (표면 형광 또는 공 촛점)의 광원을 켭니다. </li><li> 현미경을 켭니다. 현미경 용 카메라를 켭니다. 시각화를위한 현미경의 슬라이드를 넣습니다. 대물 렌즈의 개구 수를 증가시키는 침지 슬라이드에 오일을 추가한다. </li><li> 적절한 필터를 선택합니다. 초점과 적절한 목표를 조정합니다. 적절한 형광 색소를 검출하지만 광표백을 방지하기 위해 셔터를 조정한다. 샘플을 볼 때 배경 조명을 최소화하기 위해 머리 위의 조명을 유지합니다. </li><li> 변경 일E 필터 (예 DAPI 및 488 nm 인 여기 파장 용)의 다양한 형광 색소를 시각화. 카메라로 사진을 촬영합니다. </li></ol>

<ol>
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The authors declare that they have no competing financial interests.

HCV 전염병은 간경변, 간부전이나 간암의 위험에 그들을 가하고, 만성 감염을 개발 새로 감염된 환자의 80 %로, 글로벌 위협이 남아있다. 최근 두 NS3 N- 말단 단백질 분해 효소 억제제 (Boceprevir 및 Telaprevir)의 규제 당국의 승인에 의해 입증 된 바와 같이 - 직동 HCV 복제 및 성숙을 대상으로 항 바이러스제는 주요 항 HCV 에이전트를 나타냅니다. HLF 항 HCV 활성은 현재 비리 순환 그들의 타겟 간세포 내로 들어가는 것을 방지 바인딩 엔트리 바이러스 억제제로서 동작 할 것으로 생각된다. HLF에 우리의 연구는 (세포 외 비리 중화 구별) 액션의 새로운 세포 내 항 HCV기구 (7)를 제공합니다. 면역 형광을 효율적으로 생물학적 샘플에서 표적 분자의 분포 및 / 또는 위치 파악을 시각화 할 수있다. 많은 다른 형광 기법 메인 프로 중 하나에 발견면역과 관련된 문제 들는은 (형광 색소의 광 화학적 파괴) 8 photobleaching에있다. photobleaching에 의한 활동이 손실 photobleaching에 또는 강도 또는 빛의 노출 시간 범위를 줄임으로써 적은 경향이보다 강력한 형광 색소를 이용하여 중 하나를 피할 수 있습니다. 항체는 세포막을 통과 할 수 없기 때문에, 또한, 면역 형광은 일반적으로 고정 된 세포 만 제한된다. 대안 적 방법은 따라서 일반적으로 녹색 형광 단백질 (GFP) 9 형광 단백질 도메인을 포함하는 단백질의 발현에 의존 생균 단백질의 분포 / 현지화를 모니터링한다. 그러나, GFP 에피토프의 첨가는 종종 단백질 활성의 현지화 및 수정과 관련된다. 또한 면역 조직 섹션을 포함한 다른 샘플 배양 세포주, 또는 개개의 셀들의 수에 사용될 수있다 또한, 자주 모니터링하는 데 사용되는단백질, 글리 칸, 작은 분자의 지역화 / 유통. 이 기술의 주요 장점은 단순함과 형광 염색법 (예를 들어, DNA 염색)의 다른 비 - 항체 방법과 병용 할 수 있다는 사실에 상주. 또한, 상업적으로 생산 된 이차 항체는 색상의 광대 한 배열에 상대적으로 저렴하고 사용할 수 있습니다. 현재의 연구에서, HCV에게 subgenomic 플리을 지원하는 간 세포의 면역 형광은 HLF 치료시 HCV 염색 강도의 질적 저하를 한 것으로 밝혀졌습니다. 이것은 HLF의 일반적으로 받아 들여지는 약리학 적 표적이다 HCV E1 및 E2 엔벨로프 당 단백질, 부족이 subgenomic 플리 시스템과 같은 흥미있는 관측이다. 세포 외 비리 중화 활성 달리 HLF는 바이러스 복제를 저해하기 위해 HCV에 감염된 간세포에 의해 내부화 될 필요가있을 것이다. fuorescence 현미경의 사용은 우리 evalu 허용이러한 가능성을 먹었다. HCV에 감염된 간세포에 미리 배양하면, 외래 HLF 성공적으로 내면화하고, 세포 내 바이러스 복제를 저해 것으로 나타났다. 이 소설 HLF 항 바이러스 활동의 이해는이 선천성 면역 단백질의 억제의 글로벌 (그리고 가능성다면 발현 성) 메커니즘의 이해에 앞으로 단계를 구성한다.

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

흔히 면역 생물학의 여러 측면을 연구하는 데 실험 기술이다. 이것은 전형적으로 세포 및 조직에서 표적 분자의 분포 및 / 또는 위치 파악을 시각화하기 위해 사용된다. 면역은 세포 내에서 해당 항원에 대한 형광 표지 항체의 특이성에 의존한다. 모두 직접 및 간접 면역 형광 방법은 형광 색소와 링크 된 항체의 사용에 의존 할 수있다. 그것은, 낮은 신호를 제공 높은 비용과 적은 유연성을 포함하기 때문에 직접 면역 형광이 자주 사용된다. 대조적으로, 간접 면역 형광은 더 일반적 때문에 고감도의 사용과 둘 이상의 이차 항체 각 차 항체에 부착 할 수 있기 때문에 증폭 된 신호를 제공한다. 이 논문에서는, 표면 형광 현미경 및 공 초점 현미경 모두 인간 락토페린의 내재화, 면역의 중요한 구성 요소를 모니터링하는 데 사용 된간 세포로 시스템. 또한, 우리는 면역 형광을 사용하여 C 형 간염 바이러스의 복제를 HLF 세포의 억제 가능성을 모니터. 이 두 가지 접근 방식과 관련된 장점과 단점이 논의된다.

외생 투여 HLF 내부화 간 허-7 세포주의 능력에 촛점 miscroscope 면역 형광을 사용하여 모니터 하였다. HLF은 배양 배지에 첨가하고, 내재화 후, 세포 외 HLF는 PBS로 세척하고, 결합 된 잔여 HLF 막은 5 분간 트립신 처리 (1 ml)로 저하시키고, 24 시간 동안 두었다. 세포는 시드 및 면역 형광 염색하기 전에 18 시간 동안 다시 부착하는 것이 허용되었다. 세포질 한계를 개설하기 위하여, 세포 막은 488 nm의 복합체의 여기 파장의 밀 배아 응집소 (WGA) 형광 색소로 염색되었다. HLF 염색은 항 - HLF 일차 항체와 이차 항체에 연결된 633 나노 미터의 파장을 갖는 형광 색소를 사용하여 달성 하였다. HLF의 정확한 정위를 결정하기 위해, 세포를 공 초점 현미경으로 순차 0.5 μm의 수평 단면 다음 이미지화 하였다. 4 OPT를 제시 도표중간 셀 두께에 대응 iCal의 단면도. 외인성 HLF의 첨가 후, HLF 상당량은 간세포의 세포질 내부에서 검출되었다. 동일한 설정을 사용하는 수단이 없을​​ 경우에는 HLF는 미처리 세포 내부에서 관찰되지 않았다. 함께, 이러한 데이터는 간세포가 세포 외 환경에서 HLF을 획득 할 수있는 능력을 가지고 있는지 확인합니다. HCV의 세포 내 복제가 다음이었다 억제 HLF의 능력은 표면 형광 현미경을 사용하여 관찰. 복제 (HCV NS3, NS4A, NS4B, NS5A, NS5B 단백질을 발현) subgenomic HCV 레 플리 콘을지지 응-7 세포는 0, 2, 24 시간 동안 3 μM HLF 처리 하였다. HLF 및 HCV NS5A 단백질에 대한 이중 면역 염색 표면 형광 현미경으로 분석 하였다. HLF로 처리 된 세포에서 감소 하였다 (HCV 비 구조 5A 단백질의 면역 형광에 의해 측정)이 결과는 HCV 레 플리 subgenomic 염색을 나타낸다.도 5 </s<html> trong는&gt; HLF 처리 24 시간 후 HCV 염색 강도의 약 50 %의 질적 저하를 보여준다. 이것은 HLF의 일반적으로 받아 들여지는 약리학 적 표적이다 HCV E1 및 E2 엔벨로프 당 단백질, 부족이 subgenomic 플리 시스템과 같은 흥미있는 관측이다. HCV 염색 페이딩과 병용은 HLF 축적은 2 시간 후에 검출되기 시작하고, 강한 HLF 축적이 24 시간 후에 관찰 하였다. <img alt="그림 1" src="/files/ftp_upload/53053/53053fig1.jpg" /> 그림 표면 형광 현미경의 1 원칙. 현미경의 이러한 유형은, 여기 필터 (형광 표지 항체를 품고 TISSUS 또는 세포) 시료를 자극하는 특정 파장을 선택합니다. 또한, 배출 필터는 시편에 라벨을 사용하는 형광체의 발광 스펙트럼 특성과 일치하도록 선택된다.로드 / 53053 / 53053fig1large.jpg &quot;대상 =&quot;_ 빈 &quot;&gt;이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오. <img alt="그림 2" src="/files/ftp_upload/53053/53053fig2.jpg" /> 공 초점 현미경 검사의 원리는도 2. 현미경이 유형의 포커스 아웃 광 신호를 제거하기 위해 발광 핀홀을 사용한다. 초점면에 매우 가까운 형광 제조 광만을 검출 할 수 있기 때문에, 화상의 해상도는 광학 현미경 표면 형광보다 매우 우수하다. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53053/53053fig2large.jpg" target="_blank">이 도면의 확대 버전을 보려면 여기를 클릭하여주십시오.</a> <img alt="그림 3" src="/files/ftp_upload/53053/53053fig3.jpg" /> 간접 및 직접 면역 형광의 그림 3. 도식. 직접 면역 형광이 포함관심 (차 항체)의 분자에 특정한 형광 색소로 표지 된 항체의 한 유형입니다. 반면, 간접 면역 형광은 라벨이없는 일차 항체의 종류에 고유 한 형광 색소로 표지 이차 항체를 포함한다. 두 개 이상의 이차 항체가 형광 신호를 증가 각각의 일차 항체에 부착 할 수있는 것보다. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53053/53053fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 4" src="/files/ftp_upload/53053/53053fig4.jpg" /> 외생 HLF. 허-7 세포의 그림 4. 간세포의 흡수는 18 시간 동안 다시 부착시키고 면역을받는 5 분 트립신 처리에 제출, 두 번, 24 시간 동안 0 μm의 3 μm의의 HLF으로 처리 세척 하였다. 핵 승 염색플라즈마 막은 WGA (녹색 채널)과 HLF로 표지 된 i 번째 DAPI (청색 채널) 항 HLF 항체 (붉은 채널)로 염색 하였다. 이미지는 60 배의 원래 배율 공 초점 현미경에 의해 인수되었다. 수평 광학 단면은 세포의 중간 - 두께를 나타낸다. 스케일 바, 10 μm의. (11)에서 수정. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53053/53053fig4large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 5" src="/files/ftp_upload/53053/53053fig5.jpg" /> 도 5 HLF 처리 HCV 얼룩을 감소시킨다. subgenomic HCV 레 플리 콘의 복제를지지하는 어-7 세포는 0, 2, 또는 24 시간 동안 3 μM HLF로 처리하고 면역을 실시 하였다. 핵은 DAPI (파란색 채널), HCV가 안티 NS5A 항체 (적색 채널)에 의해 밝혀졌다과 HLF이 방지 HLF의 염색 한 염색 하였다ntibody (녹색 채널). 이미지는 60 배의 원래 배율 표면 형광 현미경에 의해 인수되었다. 스케일 바, 10 μm의. (11)에서 수정. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53053/53053fig5large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</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 형 간염 바이러스에 대한 인간 락토페린의 세포 흡수 및 관련 바이러스 활동을 모니터링하는 면역 형광

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

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

13.3K 조회수.  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.

비디오: 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.

인플루엔자 바이러스에 대한 높은 처리량 검출 방법

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

연구

면역학 및 감염병학

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.

Heterokaryon 형성의 두 가지 방법 HCV 제한 요소를 발견하기

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 형 간염 바이러스의 복제를 분석하기위한 프로토콜

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.

독감 조류 특정 항 체에 의해 유도 된 Fc 중재 Effector 기능을 평가 하는 방법

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

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

개발 및 검증 磁 단일 분자의 배열 디지털 효소 연결 된 Immunosorbent 분석 결과 인간 인터페론-α에 대 한

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or fluorescence microscopy. An HIV-1 reporter virus (HIV-1 Gag-iCre) is generated by inserting Cre recombinase into the HIV-1 genome between the matrix and the capsid proteins of the Gag polyprotein. This results in a packaging of Cre recombinase into virus particles, which is then released into a target cell line stably expressing a Cre recombinase-activated red fluorescent protein (RFP) to GFP switch cassette. In the basal state, this cassette expresses RFP only. Following the delivery of Cre recombinase into the target cell, the RFP, flanked by loxP sites, excises, resulting in GFP expression. This assay can be used to test any inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems and has been used to identify a class of purinergic receptor antagonists as novel inhibitors of HIV-1 viral membrane fusion.

We describe a cell-based assay to report on HIV-1 fusion via the expression of green fluorescent protein detectable by flow cytometry or fluorescence microscopy. It can be used to test inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems.

높은 처리량 Cre Lox 활성화 에이즈-1 바이러스 막 융해의 억제제를 식별 하기 위해 바이러스 막 융합 분석 결과

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or ...

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.

형광에 의한 바이러스성 RNA의 탐지<em&gt; 현장에서</em&gt; 하이브리드화 (물고기)

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

생물학

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.

바이러스 핵산 및 단백질을 시각화하고 HIV, HTLV, HBV, HCV, 지카 바이러스 및 인플루엔자 감염을 모니터링하기 위한 단일 세포 다중화 형광 이미징

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

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.

인간 거대세포바이러스의 중화를 측정하기 위한 면역형광 방법

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; ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Imaging and Visualization Techniques

A Viral Attachment Assay for Screening of Antiviral Compounds

Source: Tai, C. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/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.

항바이러스 화합물 스크리닝을 위한 바이러스 부착 분석

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