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.

이 프로토콜은 국내 및 국제 법규 및 지역 규정에 엄격한에 따라 수행되어야한다. 연구 목적으로 혈액을 기증 동의를 준 건강한 기증자로부터 혈액이 기관이 계약을 체결 한있는 수혈 센터에서 얻을 수있다. 인간의 혈액을 사용하는 경우 특별 보호는주의해야합니다. HIV-1과 실험은 3 또는 2 (BSL-3 또는 2) 연구소는 현지 법률에 따라 바이오 안전성 수준에서 수행해야합니다. 접착에 의한 밀도 구배 원심 분리 및 선택에 의해 인간 단핵구 유래 대 식세포 (hMDMs) 1. 준비 <ol><li> 건강한 기증자 (9 ㎖)에서 신선한 혈액으로 시작합니다. 70 ml의 최종 용적을 수득 칼슘 및 마그네슘 2+없이 멸균 1X 인산 완충 생리 식염수 (PBS)와 신선한 혈액의 전체 부피를 희석하고 부드럽게 개의 50 ㎖ 원뿔형 튜브에 희석 된 혈액을 추가 (튜브 당 35 mL) 중 , 15 ml의 상단에eutral가 높은 각 튜브 이미 용액에 높은 질량 친수성 ​​다당류 지형. </li><li> 브레이크없이 20 ° C에서 20 분 동안 537 XG에 혈액을 원심 분리기는 모두 튜브. 그런 계면 흐린 셀 링에 포함 된 말초 혈액 단핵 세포 (PBMC)를 수집하고 칼슘 및 마그네슘 2+없이 1X PBS 15 ml를 함유하는 새로운 50 ㎖ 튜브로 옮긴다. </li><li> 원심 분리기 20 ° C에서 5 분 동안 218 XG에 세포와 칼슘 및 마그네슘없이 1X PBS 45 ml의에서 펠렛을 재현 탁 2+. </li><li> 원심 분리는 20 ℃에서 5 분 동안 218 XG에서 세포는 칼슘 및 마그네슘 2+없이 1X PBS 10ml에 펠렛을 재현 탁하고 트리 판 블루에 1/200의 최종 희석액으로 희석하여 세포 수를 계산. </li><li> 20 ° C에서 5 분 동안 218 XG에 세포를 원심 분리기 1640 배지 2 mM의 L- 글루타민 및 100 μg의 / ㎖로 보충 된 RPMI penic (로스웰 파크 메모리얼 연구소)에 펠렛을 재현 탁illin - 스트렙토 6 웰 - 플레이트의 각 웰에서 배지 2 ㎖에 웰 당 7 × 106 PBMC를가하도록. </li><li> 2 시간 동안 5 % CO 2와 37 ° C에서 접시를 품어. 2 시간 후 단핵구는 플라스틱에 부착 된 것입니다. </li><li> 갓 절연 단핵구가 hMDMs로 분화 배지를 흡인하고 보충 2ml의 hMDM 배지 (RPMI 1640, 태아 송아지 혈청 (FCS를 decomplemented 10 %), 2 mM L- 글루타민, 1 % 페니실린 - 스트렙토 마이신)로 교체 할 수 있도록하려면 재조합 인간 대 식세포 콜로니 자극 인자 10 ng를 / ml의 최종 농도 (RHM-CSF). </li><li> 십일일 (그림 1)에 대한 5 % CO 2와 37 ° C에서 세포를 품어. </li><li> 하루에 11에서 매체를 제거하고 잘 따라 차가운 hMDM 매체의 2 ㎖로 2 회 씻는다. 다음 차가운 1X PBS 1 ml의 각 웰을 2 회 반복한다. </li><li> (2 mM의 ethylenediaminetetraacetatic 산 차가운 1X PBS 1 ml의 각 웰에 1 시간을 씻어 완전히 차별화 된 hMDMs를 분리하려면EDTA) 및 4 ℃에서 15 내지 60 분 동안 잘 당 2 mM의 EDTA와 차가운 1X PBS 2 ㎖에 세포를 배양한다. 참고 : 세포가 존재 분리하는 다른 방법 (예를 들면, 트립신 또는 트립신 같은 활동) 좋은 결과 (11)을 줄 수 있습니다. </li><li> (상하 웰 부드럽게 피펫 완료) 분리 후, 세포를 수집하고 차가운 hMDM 배지 10 ㎖를 함유하는 얼음에 50 ㎖ 튜브에 넣어. </li><li> 원심 분리는 20 ℃에서 5 분 동안 218 XG에 세포를 차가운 hMDM 배지 10ml에 펠렛을 재현 탁하고 트리 판 블루로 1/20 희석하여 세포 수를 계산. </li><li> 35mm 현미경 수준의 유리 바닥 접시 당 1 × 10 6 hMDMs에서 세포를 시드 및 1 일 5 % CO 2와 37 ° C에서 접시를 품어. </li></ol> HIV-1 바이러스 성 주식의 2. 생산 및 정량화 참고 : NLR4.3 HIV-1 개그 - iGFP (녹색 형광 단백질) M. Schindl에서 R5-열대 봉투, 선물을 들고ER 10 마크로파지를 감염 실시간으로 감염된 세포를 참조하는 데 사용된다. <ol><li> 상업 형질 전환 시약을 사용하여 해당 프로 바이러스 DNA의 6 μg의 인간 배아 신장 293T 세포 (2 × 106에서 100mm 요리)의 형질 전환에 의해 바이러스 주식을 생성합니다. </li><li> 세포 및 계수의 베타 갈 락토시다 착색 이어 바이러스 주식의 희석액을 사용하여 (HIV-1 LTR의 제어하에 베타 갈 락토시다 제 유전자를 함유) 헬라 TZM-BL 인디케이터 세포를 사용하여 바이러스 주식의 감염성 정량화 의 파란색 셀 (12)는. </li></ol> HIV-1 hMDMs 3. 감염 <ol><li> hMDMs (1.13 도금 세포)에 감염 (MOI)의 다양성에 hMDM 매체의 0.02-0.03 1 ml에 바이러스를 추가합니다. 우물 hMDM 매체의 1ml를 추가 제어하고 이일 (그림 1)에 대한 5 % CO 2와 37 ° C에서 요리를 부화합니다. </li><li> 하루에 2 hMDM 의대로 세포를 씻어3 회 IUM과 접시 당 신선한 hMDM 매체의 1ml를 추가합니다. 육일 (그림 1)에 대한 5 % CO 2와 37 ° C에서 세포를 품어. </li></ol> 양 적혈구 4. 옵 소닌 <ol><li> 접시 당 7 × 106 SRBCs의 제조를위한 4 분 동안 600 × g으로 원심 분리로 1X PBS에 0.1 % 소 혈청 알부민 (BSA)을 함유하는 용액 100 ㎕에 SRBCs 두 번 세척 하였다. </li><li> SRBCs 5 μL 당 서브 응집 농도 토끼 IgG 형 SRBCs와 PBS / 0.1 % BSA 500 ㎕의 1X에서 세정 SRBCs을 재현 탁하고 실온에서 30 분 동안 회전 배양한다. 주 : 안티 SRBCs의 IgG 서브 응집 농도를 측정 96 웰 플레이트에 20 ㎕의 1 / 25,600로 1/50에서 (13.1 ㎎ / ㎖ 농도에서 스톡)의 IgG의 희석액을 제조 하였다. 각 웰에 20 μl를 2 × 10 6 SRBCs을 추가하고 몇 시간 동안 어두운 방에서 접시를 넣어. 서브 응집 농도 인직전 잘 응집 (IgG를 + SRBCs 네트워크를 형성)로 잘 희석. </li><li> 회전 후 4 분 동안 600 XG에서의 IgG-옵 소닌 - SRBCs을 원심 분리하고 4 분 동안 600 XG에서 원심 분리하여 1X PBS / BSA 0.1 % 100 ㎕로 씻는다. </li><li> 2 mM의 L- 글루타민 및 1 % 페니실린 - 스트렙토 마이신 (1 ㎖ / 접시)이 보충 된 예비 가온 된 페놀 레드없는 RPMI 배지에서의 IgG - 옵 소닌-SRBCs를 재현 탁. </li></ol> 5. 라이브 셀 비디오 현미경 탐식의 분석 <ol><li> 이러한 CO 2의 가습을위한 물이 병을 통과하여 37 ℃의 가열 챔버를 구비 한 회전 디스크 시스템과 같은 촛점 이미징 시스템을 사용한다. </li><li> 식세포 분석의 시작 전에 37 ℃에서 현미경 스테이지를 가지고 실험 전에 가열 실 켜기. 현미경과 컴퓨터를 켜고, 및 이미징 소프트웨어를로드합니다. </li><li> 이러한 주사 속도 MAGNIFICAT로서 촬상 설정을 최적화이온, 해상도 등은 60 ~ 120 분 사이에 매 분마다 필드 당 이미지 한 프레임에 적어도 하나의 셀을 가지고 있습니다. 참고 : 여기에, 샘플 63X 렌즈와 함께 60 분 동안 하나의 프레임 분마다 이미징된다. </li><li> 현미경 무대에 영상 접시를 놓습니다. 초점을 조정하고 위치는 필드에 하나의 전체 HIV-1에 감염된 대 식세포를 찾을 수 있습니다. 사용 된 영상 시스템 및 프로브에 따라 적합한 여기 / 방출 설정을 사용합니다. phagosomes을 관찰 밝은 필드 (BF) 채널을 포함합니다 (그림 1II). 투과광 노광 시간의 비율을 조정함으로써, 상이한 채널 이미지의 모양을 최적화한다. 참고 : 여기에, NLR4.3 HIV-1 개그 - iGFP 레이저의 20 % (그림 1I)와 노출 시간의 50 밀리 초와 491 nm의 레이저를 사용하여 흥분했다. </li><li> 이미징 접시를 제거하고 접시에 7 × 10 6 SRBCs / ㎖ (그림 1)에서 SRBC 현탁액 1 ㎖를 추가합니다. </li><li> 500 XG에 원심 분리기RT에서 2 분, 식균 작용이 동기화 원심의 끝 시간을 기록하고, 상기 스테이지에 접시를 반환한다. </li><li> 적어도 1 시간 동안 매분 - (통상 20 평면 0.3 ㎛의 스텝 거리, 셀의 두께에 걸쳐) Z-스택의 초점 캡처 GFP 및 BF 이미지를 최적화한다. 사용 된 영상 시스템의 기본 파일 형식으로 시간 경과 비디오를 저장합니다. 참고 : 여기에, 시간 경과 영화가 네이티브 형식으로 저장된 * .stk 파일. </li></ol> 시간 경과 영화 6. 분석 <ol><li> 비디오 편집 메뉴 &#39;앱&#39;다운 비디오 편집 소프트웨어의 탭에 &quot;검토 다중 차원 데이터&quot;에 드롭을 클릭합니다. <ol><li> 파일을 열려면 다음 &quot;디렉토리 선택&quot;에서 &quot;선택 자료 파일&quot;을 클릭합니다. &quot;데이터가 설정&quot;상자에서 (.nd 형식)을 분석하고 &#39;보기&#39;를 클릭 인수를 선택합니다. 데이터는 열 경시 표로 표현라인에서 D의 Z-계획. </li><li> A ~ Z 프로젝션 (그림 2, 왼쪽 패널)에 감염을 표현하려면 &quot;파장&quot;상자에 491 nm 파장을 선택하고 &quot;모든면&quot;와 &quot;Z 투사&quot;탭을 클릭합니다. </li><li> (시간 순서 분석은 &quot;파장&quot;상자에 BF 파장을 선택하고 외부 SRBCs (그림 2, 빨간색 화살표), 내부 SRBCs (그림 2, 빨간색 원)과 핵을 구별하기 위해 Z 축에 최적의 평면을 선택합니다 그림 2, 파란색 원). </li><li> 비디오 몽타주을 저장하려면, &quot;선택 [X]의&quot;탭을 누른 다음 &quot;로드 이미지 (들)&quot;을 클릭합니다. 마지막으로,이 .tif 형식으로로드 된 이미지를 저장하고 ImageJ에 소프트웨어에 다음을 엽니 다. </li></ol></li><li> 핵의 위치와 BF 채널에서 관찰 된 다른 phagosomes (그림 3)을 측정하기 위해 ImageJ에 소프트웨어를 플러그인 &quot;수동 추적&quot;를 사용합니다. <ol><li> 다우ImageJ에 웹 사이트의 &quot;수동 추적&quot;플러그인을 nload. ImageJ에에서 (그림 3, 1 단계 및 2) 플러그인 및 이미지 시퀀스를 분석 할 엽니 다. </li><li> 이러한 인접 프레임 사이의 시간의 양을 나타낸다 &quot;시간 간격&quot;, 픽셀 당 거리를 나타내는 &quot;X / Y 보정&quot;(그림 3, 3 단계)로 설정을 입력합니다. 주 : 63X 크게하고 6.45 X 6.45 μm의 (2)의 화소 크기를 갖는 카메라를 사용하기 때문에 여기서, 각 프레임 및 0.205 ㎛, X / Y의 교정과 1 분의 시간 간격으로 화상 시퀀스를 저장한다. </li><li> 추적을 시작하려면 (그림 3, 4 단계) &quot;트랙 추가&quot;를 클릭하고이 내면화 될 때 제 시간에 SRBC 센터를 클릭합니다. 다음 프레임이 자동으로 나타납니다. </li><li> 시간 동안 다른 위치 (그림 3, 빨간색 상자에 6 단계)가 모든 프레임에서 SRBC 센터를 클릭하여 계속합니다. <ol><li> 그 중심을 클릭하여 모든 프레임에서의 위치를​​ 가지고 핵을 추적하기 시작합니다. 다음 시간 SRBCs의 기능에서 다른 자신의 내면화의 (프레임)에서 하나 하나 (중심을 클릭하여) phagosomes를 추적 할 수 있습니다. 편의 BF 채널에 SRBC를 참조하려면, 추적 (그림 3, 5 단계) 동안 &quot;밝기 및 명암 대비&quot;창을 사용합니다. </li></ol></li><li> 각 SRBC 추적 사이에 새로운 트랙을 가지고 (그림 3, 4 단계) &quot;트랙 추가&quot;를 클릭합니다. 추적의 수는 두 번째 열에서 변경되며, 그 결과 테이블의 &quot;트랙 N °&quot;(도 3, 단계 6). </li><li> 분석의 다음 단계를 계속하기 위해 스프레드 시트의 데이터를 저장합니다. </li></ol></li><li> (핵 및 SRBCs의 국제화 후 첫 5 분 동안 phagosomes의 속도를 향해 SRBCs를 포함하는 phagosomes의 이동 거리를 계산하기 위해 인터넷을 스프레드 시트 소프트웨어를 사용하여gure 4). <ol><li> 스프레드 시트 테이블과 새 스프레드 시트 파일을 엽니 다. 이 새로운 다음 매개 변수 전용 파일, 시간, x 축에 전송하고 핵 및 모든 SRBCs (그림 4A)의 좌표 Y-. </li><li> , 그들의 좌표 SRBCs과 핵 사이의 거리를 계산 XY 좌표 평면 (그림 4B)에서 핵 및 SRBC을 고려합니다. 주 : 두 점 사이의 거리를 연결 경로의 길이이다. 평면에서 SRBC과 핵 사이의 거리는 피타고라스의 정리에 의해 주어진다. <ol><li> C (핵과 SRBC 사이의 거리) 및 빗변의 길이를 나타내고, (B) 다른 두 변의 길이를 나타내는 경우, 피타고라스 식은 피타고라스 정리를 표현 : <img alt="식 (1)" src="/files/ftp_upload/54568/54568eq1.jpg" /> 주 : 동시에, 직교에서 수평 거리A는 (X 핵 -x SRBC)는 수직 거리 (B)는 (SRBC -y Y 핵)이다. </li><li> 따라서, 핵 및 SRBC (도 4a, 퍼플 박스)에 의해 사이의 거리를 계산한다 : <img alt="식 (2)" src="/files/ftp_upload/54568/54568eq2.jpg" /> 주 : μm의의 거리를 갖고, X / Y 교정하여 픽셀 단위로 획득 된 거리 값을 곱한다. 여기서, X / Y의 교정은 0.205 μm의 것이다. </li><li> 각 시간에서, 핵 초기 위치 (도 4C)에 SRBC 사이의 거리를 뺀다. </li><li> 처음 5 분 동안 측정 시간에 대하여 플롯. 플롯에 선형 트렌드 라인 (그림 5A)를 적용하고 SRBC 국제화 후 첫 5 분 동안 핵을 향해 phagosome 속도를 나타내는 선형 트렌드 라인의 기울기를 결정한다. </li><li> 평균 오차 통계를 계산하는 데이터를 대조속도 값을 적절한 소프트웨어 (도 5B)를 사용하여 적절한 형태로 이들을 플롯 및. </li></ol></li></ol></li></ol>

<ol>
	<li>Flannagan, R. S., Jaumouille, V., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+biology+of+phagocytosis.">The cell biology of phagocytosis.</a> Annu. Rev. Pathol. 7, 61-98 (2012).</li><li>Niedergang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Encyclopedia of Cell Biology. 2, 751-757 (2016).</li><li>Blocker, A., Griffiths, G., Olivo, J. C., Hyman, A. A., Severin, F. 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The authors have nothing to disclose.

이 기술은 몇 가지 중요한 단계가 있습니다. 감염의 백분율 도너 의존하기 때문에 우선, HIV-1에 의한 hMDMs의 제조 및 감염은 매우 중요하다. 잠재적으로 생체 내에서 바이러스에 의해 발생하는 대 식세포의 상태가 아니라 지금까지 특징되지 않았기 때문에 참고로, 우리는 이전에 감염 체외에서 편광되지 않은 대 식세포를 사용하기로 결정했습니다. 우리는 대 식세포는도없고 M1 M2임을...

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 border="0" cellpadding="0" cellspacing="0" width="691">
	<tbody>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Falcon 100mm TC-Treated Cell Culture Dish</td>
			<td style="width:124px;">Corning</td>
			<td style="width:111px;">353003</td>
			<td style="width:245px;">For viral production</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Glass Bottom Dishes 35 mm uncoated 1.5</td>
			<td style="width:124px;">MatTek corporation</td>
			<td style="width:111px;">P35G-1.5-14-C Case</td>
			<td style="width:245px;">For acquisition</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Falcon Tissue Culture Plates 6-well</td>
			<td style="width:124px;">Thermo Fischer Scientific</td>
			<td style="width:111px;">Corning. Inc. 353934</td>
			<td style="width:245px;">For human monocyte-derived macrophages</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Ficoll-Plaque PLUS</td>
			<td style="width:124px;">Dominique Dutscher</td>
			<td style="width:111px;">17-1440-03</td>
			<td style="width:245px;">a neutral, highly branched, high-mass, hydrophilic polysaccharide in solution for density centrifugation</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">DPBS, no calcium, no magnesium</td>
			<td style="width:124px;">Thermo Fischer Scientific</td>
			<td style="width:111px;">14190-094</td>
			<td style="width:245px;">Room temperature</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Dulbecco&#39;s Modified Eagle Medium (DMEM) 1X, liquid (High Glucose)</td>
			<td style="width:124px;">GIBCO, Molecular probes</td>
			<td style="width:111px;">31966-021</td>
			<td style="width:245px;">Conserved at 4&#176;C ; for HEK cells culture</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">RPMI 1640 medium GLUTAMAX&#160; Supplement</td>
			<td style="width:124px;">Life technologies</td>
			<td style="width:111px;">61870-010</td>
			<td style="width:245px;">Conserved at 4&#176;C; for hMDMs culture</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">F&#339;tal Calf Serum (FCS)&#160;</td>
			<td style="width:124px;">Eurobio</td>
			<td style="width:111px;">CVFSVF0001</td>
			<td style="width:245px;">Conserved at -20&#176;C ; decomplemented</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:124px;">Thermo Fischer Scientific</td>
			<td style="width:111px;">15140-122</td>
			<td style="width:245px;">Conserved at -20&#176;C ; for hMDMs culture</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">RPMI 1640 medium, no phenol red (10x500 ml)</td>
			<td style="width:124px;">Life technologies</td>
			<td style="width:111px;">11835-105</td>
			<td style="width:245px;">Warm in 37&#176;C water bath before use ; for phagocytosis assay</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">FuGENE6 Transfection Reagent</td>
			<td style="width:124px;">Promega</td>
			<td style="width:111px;">E2692</td>
			<td style="width:245px;">Conserved at 4&#176;C ; for viral production</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Sheep red blood cells (SRBCs)</td>
			<td style="width:124px;">Eurobio</td>
			<td style="width:111px;">DSGMTN00-0Q</td>
			<td style="width:245px;">Conserved in Alsever buffer at 4&#176;C before use</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Anti-sheep red blood cells IgG</td>
			<td style="width:124px;">MP Biomedicals</td>
			<td style="width:111px;">55806</td>
			<td style="width:245px;">Conserved at 4&#176;C</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Bovine Serum Albumin heat shock fraction, pH 7, &#8805;98%&#160;</td>
			<td style="width:124px;">Sigma</td>
			<td style="width:111px;">A7906</td>
			<td style="width:245px;">Conserved at -20&#176;C</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Inverted microscope DMI600</td>
			<td style="width:124px;">Leica</td>
			<td style="width:111px;"></td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Confocal Spinning Disk Unit&#160; CSU-X1M1</td>
			<td style="width:124px;">Yokogawa</td>
			<td style="width:111px;"></td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">491 nm 50mW laser</td>
			<td style="width:124px;">COBOLT CALYPSO</td>
			<td style="width:111px;"></td>
			<td style="width:245px;"></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">HCX PL APO CS Objectif</td>
			<td style="width:124px;">Leica</td>
			<td style="width:111px;"></td>
			<td style="width:245px;">Objective lens ; Magnification 100x ; Numerical aperture 1.40 ; Immersion oil</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">CoolSnap HQ2 (FireWire) Camera</td>
			<td style="width:124px;">Photometrics</td>
			<td style="width:111px;"></td>
			<td style="width:245px;">Pixel size 6.45 x 6.45 &#181;m ; Definition 1392 x 1040 ; Encoding the image in 14 Bit</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">Metamorph 7.7.5 software</td>
			<td style="width:124px;">Molecular Devices</td>
			<td style="width:111px;"></td>
			<td style="width:245px;">For the control of the microscope</td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:212px;">GraphPad Prism software</td>
			<td style="width:124px;"></td>
			<td style="width:111px;"></td>
			<td style="width:245px;">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 감염과 감염되지 않은 hMDMs 의해 식세포 작용의 FcR 매개 초상권 타겟으로의 IgG - 옵 소닌 SRBCs (도 1)를 사용하여 여기에서 설명된다. 이 프로토콜의 중요한 단계는 hMDMs의 준비 및 HIV-1 감염이다. 실제로, 차별화 된 대 식세포의 수율과 품질은 도너 사이 변화뿐만 아니라 10~40% 범위의 효율성과 감염율. 또한, IgG의-옵 소닌 - SRBCs의 준비는 이것이 그들의 인식?...

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 감염 라이브 차 인간 대 식세포에서 측정 Phagosome 마이그레이션 및 속도

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

Research

JoVE Journal

Immunology and Infection

8.1K 조회수.  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. 

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

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

V3의 HIV - 1 Tropism의 Genotypic 추론 사용 인구를 기반 시퀀싱

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

연구

면역학 및 감염병학

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

준비 및 사용 HIV - 1 차 감염된 CD4 + 대상 세포로 T 세포 자연 킬러 세포 세포 독성 Assays에서

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). 
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. 
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

표준 임상 Assays에 의해 탐지의 한도 이하 바이러스성로드와 HIV 감염된 개인의 확장 및 HIV - 1 RNA를 Quantifying

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

In vitro Uncoating of HIV-1 Cores

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone1, 2. Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core.
Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form3 in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells4. This indicates that the stability of the capsid is critical for HIV-1 infection.
HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37&deg;C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability4, 5, 6. It should also be useful for studying the role of cellular factors in HIV-1 uncoating.

In vitro Uncoating of HIV-1 Cores

Uncoating is an essential step in the early phase of the HIV-1 life cycle and is defined as the disassembly of the capsid shell and the release of the viral ribonucleoprotein complex (vRNP). Here, we demonstrate techniques for isolating intact cores from HIV-1 virions and for quantifying their uncoating in vitro.

HIV - 1 코어의 시험 관내 Uncoating에서

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

의 이미징 HIV-1 봉투 유발 Virological 시냅스와 합성 지질 Bilayers에 신호

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

<em&gt; 체외에서</em공부하기&gt; 공동 감염 모델<em&gt; Plasmodium falciparum</em인간의 기본 단핵구 파생 면역 세포에서&gt; - HIV-1 상호 작용

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

새로운 도구는 HIV-1에 감염된 개인으로부터 규제 T 세포를 확장하려면

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

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.

라이브 식세포에 Phagolysosome 속생의 연구

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

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1 감염 CD4의 타고난 감지 평가<sup&gt; +</sup&gt;를 사용하여 형질 수지상 세포에 의한 T 세포<em&gt; 예 생체 내</em&gt; 공동 문화 시스템.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3&#39; ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

This report describes an in vitro assay for measuring the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC)-associated integration activity in the cytoplasmic extracts of acutely infected cells. The integration of PIC-associated HIV-1 DNA into heterologous target DNA is quantified by using a nested real-time polymerase chain reaction strategy.

측정<em&gt; 체외</em&gt; HIV-1 사전 통합 단지의 통합 활동

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...