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.

该协议在严格按照国家和国际法规和地方法规来进行。从给他们的同意献血用于研究目的的健康捐献者的血液已经从与该机构签署协议，输血中心获得。特殊的保护必须用人类血液时服用。 HIV-1的实验必须在生物安全3级或2（BSL-3或2）实验室根据当地法规进行。 1.人单核细胞 - 巨噬细胞（hMDMs）通过密度梯度离心与选择附着力的制备<ol><li>开始与新鲜血液来自健康供体（9毫升）中。稀释的新鲜血液，用无菌1×磷酸盐缓冲盐水（PBS）的整个体积不含Ca 2+和Mg 2+，得到70 ml的终体积，并轻轻稀释血液加入到两个50毫升锥形管中（35毫升，每管）上15毫升的顶eutral，高度支化的，高的质量，亲水性多糖在溶液已经在每个管中。 </li><li>离心机无论是在537 XG 20分钟血管在20°C不带刹车。然后收集包含在界面处的混浊细胞环的外周血单核细胞（PBMC），并将其转移到含有15毫升1×PBS中的不含Ca 2+和Mg 2+新50ml管中。 </li><li>离心机在20℃下将细胞在218×g离心5分钟，悬浮在45ml 1×PBS中的沉淀不含Ca 2+和Mg 2+。 </li><li>离心将细胞在20℃218×g离心5分钟，悬浮在10ml 1×PBS中的沉淀不含Ca 2+和Mg 2+，以及通过稀释到1/200在锥虫蓝最终稀释计数细胞。 </li><li>离心将细胞在218×g离心5分钟，在20℃重悬在RPMI（罗斯韦尔园区纪念研究所）粒料1640培养基补充了2mM L-谷氨酰胺和100微克/毫升penicillin -链霉素具有每孔7×10 6个 PBMC中的2ml培养基中在6孔板的各孔中。 </li><li>用5％的CO 2孵育所述板在37℃下2小时。 2小时后单核细胞将已附着于塑料。 </li><li>以允许新鲜分离的单核细胞分化成hMDMs，吸出培养基并用补充用2ml HMDM培养基（RPMI 1640,10％decomplemented胎牛血清（FCS），2mM的L-谷氨酰胺和1％青霉素 - 链霉素）替换它重组人巨噬细胞集落刺激因子（RHM-CSF）在10纳克/毫升的最终浓度。 </li><li>孵育细胞在37℃，5％CO 2的11天（ 图1）。 </li><li>第11天除去培养基并用2ml每孔冷HMDM培养基洗2次。接着用1毫升冷1X PBS的清洗每孔2次。 </li><li>要分离完全分化的hMDMs，每孔1次洗1毫升冷PBS 1X与2毫米乙二胺四乙酸（EDTA）中，孵育细胞2毫升冷1×PBS中的每孔2毫摩尔EDTA，在4℃下15-60分钟。 注:替代方法来分离细胞存在（ 如胰蛋白酶或胰蛋白酶样的活动）可能得到好的结果11。 </li><li>支队（轻轻吹打向上和向下在很好地完成）后，收集细胞，并把它们放在50毫升管含10毫升冷HMDM中的冰。 </li><li>离心将细胞在20℃218×g离心5分钟，重悬沉淀在10ml冷HMDM培养基中，通过在台盼蓝稀释1/20计数细胞。 </li><li>种子的细胞以每35毫米显微镜级玻璃底培养皿1×10 6 hMDMs并在37℃用5％CO 2孵育平板1天。 </li></ol> HIV-1病毒种群2.生产和定量注:NLR4.3 HIV-1的Gag iGFP（绿色荧光蛋白）携带R5嗜信封，礼品从M. Schindl器10被用于感染的巨噬细胞，并实时看到感染细胞。 <ol><li>通过人胚肾293T细胞（2×10 6个在100mm皿），使用市售的转染试剂中的相应原病毒DNA的6微克的转染产生的病毒的股票。 </li><li>使用指示细胞的HeLa TZM-BL使用后跟细胞及数量的β-半乳糖苷着色病毒种群的连续稀释液（带有β-半乳糖苷基因的HIV-1 LTR控制下）量化病毒种群的感染性蓝小区12。 </li></ol> 3.与HIV-1 hMDMs感染<ol><li>添加病毒以感染复数（MOI）的多个0.02-0.03于1ml HMDM介质与hMDMs（镀在1.13细胞）。对照孔添加只需1毫升HMDM培养基中，并在37℃，5％CO 2的2天（ 图1）孵育碗碟。 </li><li>在第2天洗细胞与HMDM MEDIUM 3次，并添加每个培养皿新鲜HMDM介质1毫升。孵育细胞在37℃，5％CO 2的6天（ 图1）。 </li></ol> 4.绵羊红细胞的调理作用<ol><li>对于每盘7×10 6 SRBCs制备，洗SRBCs两次在100μl的1×PBS中的离心分离含有0.1％牛血清白蛋白（BSA）溶液在600 xg离心4分钟。 </li><li>悬浮洗涤SRBCs于500μl1×PBS / BSA 0.1％用兔IgG抗SRBCs以每5微升SRBCs的子凝集的浓度，并在室温与旋转温育30分钟。 注:确定抗SRBCs IgG的子凝集的浓度，（13.1毫克/毫升的库存浓度）从1/50于20μl在96孔板制备的IgG连续稀释至1 / 25,600。加2×10 6 SRBCs在20μl每孔中，并把在黑暗的房间的板几个小时期间。子凝集的浓度的之前的井用凝集抗体（IgG + SRBCs形成网络）的孔的稀释度。 </li><li>旋转后，离心的IgG调理的-SRBCs在600×g离心4分钟，并在600 xg离心用100μl的1×PBS / BSA 0.1％洗用离心4分钟。 </li><li>重悬的IgG调理的-SRBCs在补充有2mM L-谷氨酰胺和1％青霉素 - 链霉素（1毫升/皿）预热无酚红的RPMI培养基中。 </li></ol> 5.活细胞显微视频分析吞噬<ol><li>使用共焦成像系统，例如在37℃下装有加热室用CO 2穿过瓶用水加湿纺丝磁盘系统。 </li><li>之前打开所述加热室向实验吞噬测定开始前有显微镜阶段在37℃。打开显微镜和计算机，并加载图像处理软件。 </li><li>优化成像设置诸如扫描速度，尊离子，分辨率等具有每场和图像的一帧60至120分钟之间的每一分钟的至少一个细胞。 注意:在此，将样品60分钟以63X透镜期间成像的每分钟一帧。 </li><li>放置在显微镜舞台成像菜。调整焦距和位置查找只是一个整体的HIV-1在现场感染巨噬细胞。使用基于所使用的成像系统和探头适当的激发/发射设置。包括明视场（BF）通道观察吞噬小体（ 图1II）。通过调整透射光和曝光时间的百分比优化不同信道的图像的外观。 注:在这里，NLR4.3 HIV-1的Gag iGFP很兴奋使用491纳米的激光与激光的20％（ 图1I），曝光时间为50毫秒。 </li><li>取出成像盘中，在7×10 6 SRBCs / ml至菜（ 图1）中添加1毫升SRBC悬浮液。 </li><li>离心500 XG的在RT 2分钟以同步的吞噬能力，​​记录时间在该离心分离的端部和所述盘返回到阶段。 </li><li>优化的Z堆叠聚焦和捕获的GFP和BF图像（整个细胞的厚度为0.3微米的步距 - 通常是20面）每分钟至少1小时。节省时间推移的视频中所使用的成像系统的本机文件格式。 注:在这里，定时短片被保存在本机格式，* .STK文件。 </li></ol> 6.定时短片的分析<ol><li>对于视频编辑，点击下拉菜单中选择"应用程序"，并在视频编辑软件标签"审查多维数据"。 <ol><li>要打开该文件，点击"选择基本文件"，然后在"选择目录"。在"数据集"中，选择采集分析（以.nd格式），点击"查看"。的数据被表示在一个表中的列的时间ðZ-计划行。 </li><li>代表感染的Z-投影（ 如图2所示 ，左图），在"波长"框中选择491纳米波长和单击带有"所有平面"的"Z投影"选项卡。 </li><li>分析时间序列，在"波长"框选择高炉波长，并选择在Z轴的最佳平面区分外部SRBCs（ 图2，红色箭头），内部SRBCs（ 图2，红色圆圈）和细胞核（ 图2，蓝色圆圈）。 </li><li>保存视频蒙太奇，单击"选择[X]的"选项卡，然后点击"加载图像（S）"。最后，保存。TIF格式加载的图像和明年打开他们ImageJ的软件。 </li></ol></li><li>使用上的ImageJ软件插件"手动跟踪"来测量核在高炉通道中观察到的不同吞噬体（ 图3）的位置和。 <ol><li>道琼斯n载入的ImageJ的网站上的"手动跟踪"插件。上的ImageJ，打开插件和图像序列进行分析（ 图3，步骤1和2）。 </li><li>输入诸如"时间间隔"表示的相邻帧之间的时间量，而"x / y的校准"，这表示每像素的距离（ 图3，步骤3）的设置。 注:在这里，保存图像序列与每一帧和0.205微米X / Y校正之间1分钟的时间间隔，因为63X变焦和6.45点¯x6.45微米2的像素大小相机使用。 </li><li>要开始跟踪，点击"添加轨道"（ 图3，第4步），当它是内在的第一次点击一个SRBC中心。在下一帧自动出现。 </li><li>继续点击在所有帧的SRBC中心有在时间不同位置（ 图3，步骤6中的红色框）。 <ol><li>开始跟踪核通过点击其中心有它在所有帧中的位置。下一页（点击其中心），他们当时的内化（架），在SRBCs功能不同逐个跟踪吞噬体。为方便起见，以查看高炉通道SRBC，跟踪（ 图3，步骤5）中使用的"亮度和对比度"的窗口。 </li></ol></li><li>每个SRBC跟踪之间，点击"添加轨道"（ 图3，步骤4）有一个新的轨道。跟踪的数量将在第二列被改变，结果表中的"跟踪N°"（ 图3，步骤6）。 </li><li>保存在电子表格中的数据，以继续分析的下一个步骤。 </li></ol></li><li>使用电子表格软件来计算包含对核和吞噬体的SRBCs的内化后的第5分钟时的速度SRBCs吞噬体的行驶距离（ 连接古尔4）。 <ol><li>打开电子表格表和一个新的电子表格文件。转移到这个新的文件只有以下参数，时间，x和细胞核和所有SRBCs（ 图4A）的y轴坐标。 </li><li>计算SRBCs和细胞核之间的距离，只有它们的坐标，考虑核和SRBC在XY坐标平面（ 图4B）。 注:两点之间的距离是连接它们的路径的长度。在飞机上，SRBC和细胞核之间的距离由勾股定理给出。 <ol><li>如果c（细胞核和SRBC之间的距离）表示斜边和一个的长度和b表示其他两个边的长度，表达毕达哥拉斯定理如毕达哥拉斯方程: <img alt="式（1）" src="/files/ftp_upload/54568/54568eq1.jpg" /> 注:同时，在正交的水平距离一为（x 核 -x SRBC）和垂直距离b是（γ 核 -y SRBC）。 </li><li>因此，计算出核和SRBC（ 图4A，紫色盒）通过之间的距离: <img alt="公式（2）" src="/files/ftp_upload/54568/54568eq2.jpg" /> 注:乘以所获得的距离值（以像素为单位）由X / Y校准具有在微米的距离。在这里，X / Y校正是0.205微米。 </li><li>在每个时间，减去细胞核与SRBC的初始距离（ 图4C）之间的距离。 </li><li>绘制对时间的测量的前5分钟。应用线性趋势线（ 图5A）的情节，并确定线性趋势线表示SRBC内化后的第5分钟期间向核吞噬体速度的斜率。 </li><li>整理的数据来计算的平均值和统计误差使用适当的软件（ 图5B）的速度值，并绘制它们以适当的形式。 </li></ol></li></ol></li></ol>

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The authors have nothing to disclose.

这种技术有几个关键步骤。首先，hMDMs的制备及其感染HIV-1是至关重要的，因为感染的比例捐助依赖。值得注意的是，我们决定使用未在体外感染前极化巨噬细胞，因为由体内病毒可能遇到的巨噬细胞的状态没有得到很好的迄今表征。我们检查了几种表面标记的表达，表明巨噬细胞既不M1也不M2和已验证的CD4和CCR5的表达。感染率是可变的，从10％至40％，有时低的范围内。这个变量感染率就是为什么它可能很难找到在显微镜下的HIV-1感染的荧光HMDM。此外，为了在离心步骤之后立即找到一个HIV-1感染的细胞所需要的时间也可能导致延迟在开始采集。吞噬作用可以在没有synchronizatio发起结合并通过离心产生的吸氮量。然而，没有选择此选项到达细胞前漂浮SRBCs。这可能是用珠子一个不错的选择。尽量减少找到感染细胞所需要的时间，网格玻璃底菜可以用来进行到启动吞噬作用测定法之前识别HIV-1感染hMDMs。有周围SRBCs的"合理"数量选定HIV感染的细胞将被迅速地选择启动收购。 "合理SRBC量"是指足有每个单元至少10 SRBCs并没有太多的防止外部SRBCs掩蔽的吞噬体。 此外，它以优化调理作用条件，以确保将有有效的识别和摄取的细胞是重要的。 FCR的介导的吞噬作用是组成和hMDMs是经常在FCR介导的摄取非常有效的。此外，对于正确的吞噬能力，​​条件也要到bË最优与加热室中于37℃和5％的CO 2。有必要在开始吞噬作用测定法之前，检查气体和温度。 最后外部SRBCs的从内部吞噬体的鉴别，并在高炉通道细胞核的鉴定是对的ImageJ软件分析重要。这就是为什么该通道的设置是非常重要的。鉴于重点可采集过程中丢失，我​​们建议收购Z轴图像的堆栈，以确保拥有最佳的飞机来分析吞噬小体的速度。优选有一个单一的细胞在一个场来分析每个小区内的所有内在SRBCs。这就是为什么在透镜的选择是重要的，在这里，我们使用2透镜作为细胞大小的一个函数。这是我们更容易追踪与100X镜头吞噬体，但有时细胞过大，所以我们不得不切换到63X镜头。想着IM时，镜头的选择也很关键使用ImageJ和电子表格表，因为两个镜头牵连不同的X / Y校正年龄分析。 这种技术的第一个限制是需要被获取的具有统计学显著结果的样本数。我们可以注册每个供体几部电影，但每情况通常不超过2个单元，每个菜。此限制的解决方案，以增加数目的样本，重复实验以更捐赠者或取决于显微镜做多点的XY成像。最后一个方法的不便是重点机动XYZ扫描过程中的潜在损失。另一个限制是，相对于一个自动分析速度的手动量化是冗长的。对于全自动化分析，就必须使用荧光标记的粒子，如开拓性的研究4耦合到鱼皮明胶乳胶珠的随访与MATLAB软件的跟踪算法报道。荧光标记opsonizing抗体或小珠可以是一种选择，但它们受到漂白，1小时的记录吞噬时，这是一个问题。其他的研究报告使用乳胶珠13,14。然而，我们注意到的乳胶颗粒内化比SRBCs的摄取不容易发现。的确细胞内迁移的动力学的出发点是重要的是知道检测吞噬体运动的早期缺陷。 这里所描述的方法的主要优点是它的简单性和使用自由软件。使用ImageJ的软件执行手动跟踪，我们必须使用吞噬体与细胞核的位置，因此我们需要使用毕达哥拉斯定理计算吞噬体和细胞核之间的距离，和有之后的速度在电子表格软件的线性回归。这3个步骤的分析可以降低到一个步骤，跟踪，在结冰的软件与＆＃34;手动跟踪"插件输出数据然后直接在粒子的速度跟随，并且更具体地2组（如吞噬体和核）之间的相对速度。最后，计算允许我们测量的相对速度。一个颗粒相比另一个粒子其也可移动。 这个生活时间推移分析的潜在的未来应用可以是用于颗粒在明场可见的，如细菌，真菌，或细胞碎片。不同的治疗或足以修改吞噬体成熟和巨噬细胞的活化性质将从朝向小区中心吞噬体运动的这种精制分析受益感染的条件。

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.

由HIV-1感染和非感染hMDMs的FcR介导的吞噬作用，这里描述了使用的IgG调理的SRBCs作为模型目标（ 图1）。此协议的关键步骤是hMDMs编制和感染HIV-1。事实上，差异化的巨噬细胞的产量和质量捐助者之间的差异，以及与在10-40％的范围内效率感染率。此外，IgG的调理-SRBCs的制备也很重要，以避免损坏红细胞，因为这可能导致它们的识别和摄取作为碎片，而不是通过的FcR介导的吞噬作用。最佳的调理作用将确保高效的吞噬作用。 在活的HIV感染的巨噬细胞吞噬体运动在实时与在BSL-3实验室一个旋转盘共聚焦显微镜对BF信道进行分析。高炉通道的设置是关键ŤÓ看到细胞核（ 图2，蓝色圆圈）和用于鉴别从内在SRBCs（ 图2，红色圆圈）的外部（ 图2，红色箭头）。高炉显微镜被认为是最简单的光学显微镜的照明技术足以看到吞噬体，这是比外部SRBCs少折射力。 采集图像序列后的第一个步骤是对的ImageJ软件手动跟踪（ 图3）。这一点可能特别长和繁琐的，因为它是优选由一个为所有的图像序列来跟踪每吞噬体中的一个而可以认为实验需要与每种条件至少3个供体进行多次，以达到一个足够大的样本大小统计分析（至少30吞噬体与3个不同的供体）。 第二步骤是分析在电子表格软件，以内部为每个内在SRBCs（ 图4）之后的第一个5分钟内计算吞噬体速度。对于这一点，我们绘制每个吞噬体对时间的前5分钟内的行驶距离。的代表性示例示于上图5A非感染hMDMs。接下来，我们获得吞噬体，它是线性回归曲线的斜率的速度。的0.5384微米/分钟的速度被发现的这个吞噬。 值得注意的是，它是可能的，或者以后通过在较长的时间尺度分析速度内化后的最初几分钟期间分析吞噬体速度，如这里或在迪马等人 8说明。在我们的研究中，我们观察到，在艾滋病毒感染hMDMs化后的第一个5分钟内吞噬体速度相比降低非感染hMDMs（ 图5B）。 图1的控制和对吞噬成像的HIV感染hMDMs的制备单核细胞是通过粘接法（1）和分化成巨噬细胞11天用M-CSF（2）。接着，hMDMs感染NLR4.3的HIV-1Gag-iGFPⅰ）在以MOI 0.02和0.03之间8天与第2天的洗涤（3）。 （4）IgG的调理SRBCs的1小时期间添加到hMDMs的吞噬作用。含SRBCs的吞噬小体是对BF的图像检测（红圈，二）。图片均来自施维雅图像数据库。酒吧= 10微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54568/54568fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54568/54568fig2.jpg" /> 在图2的实时采集吞噬作用由代表控制和HIV感染的hMDMs。hMDMs制备和感染如在图1中描述，与491纳米的激光检测该NLR4.3的HIV-1Gag-iGFP感染的细胞。旋转盘共聚焦图像中的Z-堆栈获得并同时使用显微镜采集软件和ImageJ的（左小图）进行分析。高炉图像画廊代表非感染表示为hh（顶部对应于电影1面板）和45分钟采集期间电池（46图像随时间的NLR4.3的HIV-1Gag-iGFP感染（相应于电影2底壁板） :毫米）。细胞膜（黑色虚线），细胞核（蓝圈），外部SRBCs（其中一些显示为红色箭头）和内部SRBCs（其中一些显示为红色圆圈）都在BF图像检测。酒吧= 10微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54568/54568fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54568/54568fig3.jpg" /> 图 3. 与ImageJ的手动跟踪插件图像分析教学步骤。在BF通道（2）打开"手动跟踪"插件（1）和加载时间推移视频后，输入采集（3）的参数。点击"添加轨道"（4），并通过点击在一个吞噬小体（5），以获得一个新的窗口与所选吞噬（6，红色框）的X和Y位置开始跟踪测量。要遵循方便的时间顺序吞噬，改变亮度和对比度（5&#39;）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54568/54568fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54568/54568fig4.jpg" /> 数字<html> 4.在电子表格软件吞噬体偏移速度的数据分析教学步骤。（A）装配在一个数据表X和细胞核的Y位置，每个吞噬（红色框）。在另一列（紫色框），计算吞噬体与毕达哥拉斯定理细胞核其中c表示核与SRBC和一之间和b直角三角形的其他两个边的长度的长度（间的距离B）。 （C）最后，通过在时间0减去SRBC和细胞核从初始距离给定的时间之间的距离，计算出每个时间（绿盒）在由吞噬体移动的距离<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54568/54568fig4large.jpg" target="_blank">，请点击此处查看大图这个数字。</a> p_upload / 54568 / 54568fig5.jpg"/&gt; 图5. 定量控制和艾滋病病毒感染hMDMs吞噬迁移速度。（A）对于每个SRBC和SRBC只是内部后，对核行驶距离被测量并绘制了时间。前5分钟，在其速度在电子表格软件利用线性回归计算。代表性的实验显示（ 图3-4的内部SRBC）。 （B）前5分钟内所有速度是在非感染（5个供体）66吞噬体和吞噬体60在HIV感染的细胞（4捐助者）进行测量。数据表示平均值±SEM（与韦尔奇的修正非配对t检验; **，P &lt;0.01）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54568/54568fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54568/Jove_movie1.mov" target="_blank">电影1:在一个代表性的非感染的巨噬细胞吞噬体的运动。 （右击下载）</a> ，用每分钟一个图像（:毫米46的图 ​​像随时间指示为11H）吞噬作用的45分钟期间在高炉信道检测到的调理红血细胞的移动。棒= 10微米。 <img alt="电影2" src="/files/ftp_upload/54568/54568movie2.jpg" /> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54568/Jove_movie2.mov" target="_blank">电影2:在有代表性的感染艾滋病毒的巨噬细胞吞噬体运动。 （点击下载）。</a> HIV-1（NLR4.3 HIV-1加格iGFP）感染的巨噬细胞与491激光照射后确定。期间45高炉通道检测调理红血细胞的运动每分钟一个图像（:46毫米带图像显示的时间为HH）吞噬分钟。棒= 10微米。

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.

吞噬体偏移和速度与HIV-1感染的活小学的人巨噬细胞测量

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

8.1K 次观看.  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. 

视频: 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.

V3的HIV - 1取向的基因型推理使用人口为基础的测序

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 ...

科研

免疫与感染

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.

制备和使用艾滋病毒感染主的CD4 + T细胞作为靶细胞，自然杀伤细胞的细胞毒性检测

 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.

在艾滋病毒感染者病毒载量低于检测限制标准的临床实验放大和量化HIV - 1 RNA

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.

在体外对HIV - 1核心脱壳

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.

艾滋病毒的成像1信封引起的病毒学突触信号合成脂双层

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.

一个<em&gt;在体外</em&gt;共感染模型研究<em&gt;恶性疟原虫</em人原发性单核细胞源性的免疫细胞-HIV-1相互作用

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.

新工具展开调节性T细胞的HIV-1感染的个体

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.

在现场研究巨噬细胞吞噬溶酶体生源

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.

评估的HIV-1感染的CD4的先天传感<sup&gt; +</sup&gt;性T细胞浆细胞样树突细胞使用<em&gt;体外</em&gt;共培养体系。

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.

测量<em&gt;体外</em&gt; HIV-1 Preintegration配合物的整合活动

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 ...