Name: phagosome migration and velocity measured in live primary human macrophages infected with hiv-1
Uploaded: 2016-09-05T15:00:00.000+00:00
Duration: 7 min 32 s
Description: Watch this Scientific Journal Video about Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1 at JoVE.com

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

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

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

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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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

Universidad San Sebastian

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

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

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

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

可视化的细胞与细胞转移的艾滋病毒，艾滋病毒和现场共聚焦显微镜的荧光克隆

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

科研

免疫与感染

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

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

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

活细胞视频显微镜病病原菌的吞噬作用

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

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

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 ...

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

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

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

高通量荧光技术巨噬细胞吞噬功能和肌动蛋白聚合的评估

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

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force for plasma membrane extension around the particle. In addition, efficient engulfment of large material relies on focal exocytosis of intracellular compartments. This process is highly dynamic and numerous molecular players have been described to have a role during phagocytic cup formation. The precise regulation in time and space of all of these molecules, however, remains elusive. In addition, the last step of phagosome closure has been very difficult to observe because inhibition by RNA interference or dominant negative mutants often results in stalled phagocytic cup formation. 
We have set up a dedicated experimental approach using total internal reflection fluorescence microscopy (TIRFM) combined with epifluorescence to monitor step by step the extension of pseudopods and their tips in a phagosome growing around a particle loosely bound to a coverslip. This method allows us to observe, with high resolution the very tips of the pseudopods and their fusion during closure of the phagosome in living cells for two different fluorescently tagged proteins at the same time.

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM

We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

"吞噬体封闭试验"可视化吞噬体形成的三个维度利用全内反射荧光显微镜（TIRFM）

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

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

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

小鼠巨噬细胞吞噬的延时3D 成像

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

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

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

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

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

使用共聚焦显微镜研究 利什曼原虫 的吞噬作用

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

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

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

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

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

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

Real-Time Imaging of Macrophage Phagocytosis of IgG-Opsonized Red Blood Cells

Source: Horsthemke, M., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/57566">Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages.</a> J. Vis. Exp. (2018)
This video demonstrates an assay to monitor independent macrophage-mediated red blood cell (RBC) phagocytosis events. Mouse immunoglobulin G (IgG)-opsonized human RBCs are incubated with mouse macrophages and observed under a confocal microscope. The RBCs are engulfed by macrophages via a phagocytic cup formation and targeted for lysosomal degradation.

IgG 调导红细胞巨噬细胞吞噬作用的实时成像

Source: Horsthemke, M., et al. Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages. J. Vis. Exp. (2018)
This video demonstrates an assay to monitor ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

TIRF Microscopy-Based Visualization of Phagosome Formation and Closure

Source: Marie-Ana&#239;s, F., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/54470">&#34;Phagosome Closure Assay&#34; to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM).</a> J. Vis. Exp. (2016).
This video demonstrates a high-resolution total internal reflection fluorescence (TIRF) microscopy technique for real-time visualization of phagosome formation and closure during macrophage-mediated phagocytosis of IgG-opsonized red blood cells (RBCs) attached to the surface of a glass bottom dish. The macrophages extend pseudopodia around the RBCs and engulf them inside the phagosomes, detaching them from the glass surface.

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

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可视化的细胞与细胞转移的艾滋病毒，艾滋病毒和现场共聚焦显微镜的荧光克隆

活细胞视频显微镜病病原菌的吞噬作用

在现场研究巨噬细胞吞噬溶酶体生源

高通量荧光技术巨噬细胞吞噬功能和肌动蛋白聚合的评估

"吞噬体封闭试验"可视化吞噬体形成的三个维度利用全内反射荧光显微镜（TIRFM）

小鼠巨噬细胞吞噬的延时3D 成像

使用共聚焦显微镜研究利什曼原虫的吞噬作用

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

IgG 调导红细胞巨噬细胞吞噬作用的实时成像

基于 TIRF 显微镜的吞噬体形成和闭合可视化

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

摘要

探索更多视频

此视频中的章节

可视化的细胞与细胞转移的艾滋病毒，艾滋病毒和现场共聚焦显微镜的荧光克隆

活细胞视频显微镜病病原菌的吞噬作用

在现场研究巨噬细胞吞噬溶酶体生源

高通量荧光技术巨噬细胞吞噬功能和肌动蛋白聚合的评估

"吞噬体封闭试验"可视化吞噬体形成的三个维度利用全内反射荧光显微镜（TIRFM）

小鼠巨噬细胞吞噬的延时3D 成像

使用共聚焦显微镜研究 利什曼原虫 的吞噬作用

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

IgG 调导红细胞巨噬细胞吞噬作用的实时成像

基于 TIRF 显微镜的吞噬体形成和闭合可视化

使用共聚焦显微镜研究利什曼原虫的吞噬作用