The authors would like to thank the following funding sources: RO1 DA 12104, RO1 DA 022935, RO1 DA031202, K05DA033881, P50 DA 011806, 1R01DA034582 (to S.R) and F31 DA026264-01A1, T32 DA07097 (to J.N.).

注意:此协议可以如在我们先前发表的作品12使用用于多种应用，如吞噬作用和肌动蛋白聚合的定量。的协议适合于各种变形例， 表1列出的细胞类型和颗粒在过去的研究中成功地使用。规范使用该协议的吞噬作用或肌动蛋白聚合的定量说明了图1。 <img alt="图1" fo:content-width="4in" src="/files/ftp_upload/52195/52195diagram1.jpg" width="600" /> 图1:荧光分析对吞噬作用和肌动蛋白聚合的量化的图后，将细胞铺板，处理，并使其附着，标有绿色荧光素（FITC）的粒子加入到细胞吞噬作用。反应通过锥虫或antibi停止耳部清洗（以排除非内化的颗粒），并固定在4％多聚甲醛进行。细胞随后用红色荧光标记的肌动蛋白（罗丹明鬼笔）和蓝色荧光标记（DAPI）。吞噬作用和肌动蛋白聚合的索引被量化为绿/蓝（FITC / DAPI），或红/蓝（罗丹明/ DAPI）荧光的相对荧光单位的比率。 1.电镀和细胞治疗注:在开始之前，考虑与至少4技术重复运行的治疗，并包括在板布局以下控件:只有细胞（未染色的，单独或DAPI罗丹明单独）只和粒子。 <ol><li>开展1-4在无菌细胞培养罩，以防止样品污染的措施都加入试剂。 </li><li>板的巨噬细胞（鼠J774细胞系）在一个96孔板在1-5×10 4个细胞在50μl（每孔）<html>为辅预热媒体。理想板此方法是黑色的96孔板用透明或黑色的塔底，取决于读者的能力。 <ol><li>用于补充培养基，用10％热灭活的FBS，以避免补体级联的干扰，和1％青霉素/链霉素（如果不使用活细菌吞噬作用）。如果使用贴壁细胞允许1-2小时用于粘附，并用于非粘附细胞，降速板与细胞在​​500×g 10分钟。 </li></ol></li><li>在50μl中添加期望的激动剂/拮抗剂或车辆控制在2倍浓度（悬浮于PBS或更优选，在补充培养基）为100微升（1×）的最终体积并孵育时间所需量。多道移液器和试剂水库将有利于最快的处理。 </li></ol> 2.荧光体颗粒的调理作用ftp_upload / 52195 / 52195table1.jpg"WIDTH ="500"/&gt; 表1:测试的细胞类型并在荧光分析荧光颗粒吞噬作用和肌动蛋白聚合的表1示出的细胞类型的组合（细胞系和原代细胞），我们的小组通过荧光法使用。除了看吞噬作用和肌动蛋白聚合由野生型细胞中，我们还利用一些这些颗粒用于通过转染的细胞表达GFP（绿色荧光蛋白）检查吞噬。用含有荧光报告质粒的细胞的荧光，也可以用作除了DAPI 表说明一个细胞标记:A -肌动蛋白聚合; P - 吞噬; PT - 吞噬了GFP标记的转染细胞; OPDex - 调理葡聚糖珠; HKOP - 热杀调理。 <ol><li>确保荧光颗粒和标签避光在任何时候，杜尔为了防止光致漂白，以及在孵育荷兰国际集团的处理，洗涤<ol><li>对于干颗粒，称出荧光热灭活调理（HKOP）菌（E2861: 大肠杆菌 K-12株），使用制造商的规格。小心确保质量占10:1 - 20:1的细菌:细胞比率（或至少10:1的比率-在吞噬量化常用）13,14。可以由制造商提供，或者经由couting为1mg / ml储备液的连续稀释测定颗粒的数目。 </li><li>悬浮在50微升PBS，涡旋在最高设置1分钟。确保适当的涡流（在这里和在整个协议）作为颗粒倾向于聚集可能影响调理作用的功效。 </li><li>为粒子在悬浮液中，如荧光标记的葡聚糖珠（DEX珠），涡流原液1分钟，取50μl的悬浮液，或根据对在卷D的胎圈浓度珠etermine安装方便10:1珠子:细胞比例。 </li></ol></li><li>添加opsonizing试剂等体积的粒子，大力旋涡振荡为一分钟。 </li><li>与opsonizing试剂在37℃下1小时孵育颗粒。 </li><li>继温育后，在500×g离心离心颗粒5分钟，并除去含有过量opsonizing试剂的上清。通过加入100微升的PBS洗涤颗粒，涡旋重悬沉淀，并离心5分钟，在500×g下。 <ol><li>重复这些步骤两次，以确保正确的调理作用，并除去未结合的抗体。 </li></ol></li><li>通过重新悬浮它们的5ml培养基（足够一个96孔板）制备调理粒子的工作溶液，并存储避光直到除细胞。 </li></ol> 3.吞噬<ol><li>放置颗粒在无菌试剂槽的工作溶液。从这里出发，加5056;升调理粒子在步骤1（对于50毫微克/ ml的终浓度）中制备的细胞，并允许细胞吞噬发生在标准细胞培养条件。下面所示是吞噬的两种方法，可使用该方法（ 图2）来评估。 </li></ol><img alt="图2" fo:content-width="4in" src="/files/ftp_upload/52195/52195diagram2.jpg" width="600" /> 图2:连续与同步吞噬比较连续吞噬表示颗粒随时间的连续的内化，因为它们慢慢到达细胞上的井的底部。甲对比同步步骤（离心）迫使粒子到沉底，提高与细胞的颗粒接触，并导致立即的内化的细胞。同步吞噬是一个更快的过程，更迅速内化的一部分icles由于增加的细胞:颗粒接触。 <ol><li><ol><li>对于连续吞噬方法（图2），允许细胞以不断吞噬调理细菌作为它们慢慢沉底和进来与细胞接触时，利用较长的时间帧，以允许所述细菌到达井的底部，并获得在与细胞接触。如果进行一个时间过程实验，在不同时间间隔添加细菌和停止反应的整个板的同时，提高处理速度。 </li><li>对于同步吞噬方法（图2），可提高颗粒:通过离心细胞接触，降速含有细胞和颗粒（5分钟，在500×g离心）只要颗粒加入，以促进所有时间点的同步整板和加速粒子:细胞相互作用。 <ol><li>测量时间过程完成CENTRI后右起fugation一步。如果导通的时间过程实验，如下所述独立地停止各时间点在不同的时间间隔。 </li></ol></li></ol></li></ol>注:同步吞噬方法是与前一个较为费力，但在较短的时间提供了更高的吞噬指数。 4.停止吞噬<ol><li>对于贴壁细胞，除去上清液，并添加50微升稀释台盼蓝的PBS（1:2稀释）中，为了扑灭非内化的细菌的荧光。 </li><li>对于非粘附细胞，降速板并除去上清液。加入50微升稀释台盼蓝。确保到离心机每次洗涤之间的板上于（在500×g离心5-10分钟），以便最大限度地减少细胞的损失和使足够的洗涤。 </li><li>以下锥虫孵育，洗涤细胞2次，用PBS以除去任何残留锥虫（直到PBS除去从井变为清楚）。 TRypan已经显示以猝灭未内在化的荧光标记，热灭活，细菌颗粒的荧光15,16，但不适用于活细菌或OPDex珠。活的细菌，包括使用抗生素，如青霉素，链霉素，庆大霉素或代替的锥虫洗涤，以避免荧光的非内化颗粒混杂影响洗涤。 </li><li>固定的细胞用100μl的4％多聚甲醛，并孵育15分钟，在室温下，然后用PBS（以每孔100μl的每次洗涤）。在此步骤中，保存的细胞在4℃下进行长达一周，或直接进行染色和定量。 注意:所有的后续步骤，形成该点序可以在细胞培养罩的外面来完成。 </li><li>删除所有的液体，并添加50微升的DAPI的重组在PBS 5毫微克/毫升。允许5分钟进行染色，然后用PBS洗两次。在洗涤步骤中，加入50微升PBS至每个孔，并记录吞噬作用。 </l我&gt; </ol> 5.肌动蛋白染色注意:除了吞噬作用，荧光法将使肌动蛋白聚合的量化通过测定肌动蛋白，这是抑制片状伪足，pseudopodiae期间减少的强度，并且膜皱裂，与抑制剂（ 图1A）处理的结果。这是，如果除了吞噬感兴趣的肌动蛋白聚合的量化可选步骤。如果肌动蛋白聚合是最终目标，使用荧光颗粒吞噬作用是可选的。 <ol><li>下列的多聚甲醛固定（ 步骤4.3），用PBS洗涤平板两次，每孔100μl每次洗涤。加50微升0.1％的Triton X-100的PBS透化对孵育在室温-5分钟。 </li><li>用PBS洗出0.1％的Triton X-100的2倍（如上述），并用1％牛血清白蛋白（BSA）20分钟阻断，以避免拉贝的非特异性结合升。 </li><li>封闭后，取出BSA解决方案，并立即加入罗丹明鬼笔。为了制备罗丹明染色的工作溶液中，在5毫升PBS（足够用于一个板）的使用100微升methanoic溶液（由生产商提供）。从工作溶液中添加50微升每孔，并孵育20分钟，在室温下进行。 注:继洗涤步骤和DAPI染色如在步骤4.4的细胞将准备定量。 </li></ol> 6.定量<ol><li>用在λ= 355 nm，发射滤波器激发滤光片在λ= 460nm的量化使用荧光板读数器，记录绿色荧光使用激发滤光片在λ= 488 nm和发射滤波器的吞噬在λ= 5​​18纳米，和蓝色荧光。 </li><li>以通过λ= 355纳米和λ= 460nm的（如上），和红色感量化使用荧光板读数器，记录蓝色荧光的肌动蛋白聚合通过激发滤光片orescence在λ= 5​​84 nm，发射滤波器λ= 620纳米。 注:轨道或中心的检测是可以接受的记录。某些类型的细胞收集更多到阱比中心，在这种情况下，轨道记录可以是优势的边缘。特别是，平均大约轨道三个点也可以被用于获得更准确的结果。 </li><li>划分的绿色或红色荧光由蓝色荧光读数（来自DAPI）总RFU，根据粒子标签或吞噬与肌动蛋白定量（ 图1）。由于高的板到板方差，使用相对指标（如相对于T = 0的控制），以最好地说明观察到的趋势，并促进更可靠的统计分析。 </li></ol>

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The authors have no competing financial interests.

所述荧光技术的主要限制是实验方差以及与广泛洗涤和使用非粘附细胞的细胞相关的损失。 方差很大程度上观察到由于在荧光发色粒子作为制备储液的过程中称重和分注的变化不保持从实验的颗粒进行实验的相同数量的精确方式。为了解决实验之间方差的问题，数据可以表示为相对术语，例如，作为％吞噬作用或折叠从控制变更（控制 - T = 0;加入和除去颗粒立即）。这种形?...

荧光信号的量化已被广泛应用于许多科学的方法，从PCR检测，流式细胞术，激光共聚焦显微镜和FRET分析复ELISA分析。荧光成像和定量有着广泛的应用，并且可针对各种细胞过程的定量分析的一大利器。使用的荧光标记物和它们的信号已经彻底改变了在过去十年中，并且荧光板阅读器的出现促进了荧光期间的细胞过程发出的光的高通量定量。 荧光分析可以作为吞噬量化一个伟大的工具。由于吞噬细胞的发现通过Metchnikoff在1800 1吞噬了研究。多年来，各种方法已被用于研究这个重要过程抵御入侵细菌，病毒，真菌和寄生虫病原体2-5先天免疫防御必需。量化利用显微镜和体视技术，以以前的方法以可视化的细胞被吞噬，然后将其通过计算内化颗粒（手动或使用利用软件）6-8的定量。一些缺点单独使用显微镜进行定量分析是细菌人工计数是劳动密集，更容易观察偏见。在吞噬的量化使用的另一种方法是从细胞裂解物（以下吞噬）上的细菌培养板细菌电镀的微生物技术，但这种方法可能会失败占杀菌机制和部分内在的细菌的存在。相比显微镜此方法甚至更多的劳动密集和需要几天的时间来分析。流式细胞术似乎是量化吞噬的最快，最有效的方法，并已用于通过许多组9-11，但成本高，通常与在相关联的所需的分析仪器使得它的最昂贵的方法时，相比于前面提到的测定。 该荧光法是一个很好的替代流式细胞仪粒子内部的分析，因为它提供了用荧光设备偏见的量化，是不是因为成本太高。荧光的其它额外的好处是效率高，高吞吐量能力用于定量荧光的标记的内化的颗粒排出。 荧光的好处是可以外推到比吞噬其它过程定量。例如，荧光分析，可用于研究任何导致肌动蛋白聚合的变化在细胞内或膜结合的受体，改变细胞通透性/生存力，转染效率，以及调制的表达过程。荧光技术的一个缺点是，根据所使用的标签，可能有一个高实验实验变异性可通常通过证明通过相对定量数据，如倍数变化或从控制增加的百分比来解决。

<table><tbody><tr><td>1 &amp;mu;m yellow-green fluorescent Fluo-Spheres;</td><td>Molecular Probes</td><td>F8852</td><td>combine 50&amp;mu;l with 50&amp;mu;l opsonizing reagent for 30 min at 37&lt;sup&gt;o&lt;/sup&gt;C before use</td></tr><tr><td>Heat killed &lt;em&gt;E. coli&lt;/em&gt; BioParticles fluorescein conjugate</td><td>Molecular Probes</td><td>E2861</td><td>reconstitute (5 mg) in 50 &amp;mu;l of PBS and combine with 50 &amp;mu;l opsonizing reagent for 30 min at 37 &amp;deg;C before use</td></tr><tr><td>Opsonizing reagent</td><td>Molecular Probes</td><td>E2870</td><td></td></tr><tr><td>Rhodamine phalloidin</td><td>Molecular Probes</td><td>R415</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542</td><td></td></tr><tr><td>Trypan blue</td><td>Gibco</td><td>15250-061</td><td></td></tr><tr><td colspan="4">The items below are available in many brands but the items we used in this study are from the following manufacturers:</td></tr><tr><td>RPMI Cell growth media</td><td>Gibco</td><td></td><td>Supplemented with 10% FBS and 1% pennicillin/streptomycin; warm in 37 &amp;deg;C before use</td></tr><tr><td>Fluorescent plate reader - Fluostar Omega</td><td>BMG Labtech</td><td></td><td></td></tr><tr><td>Paraformaldehyde (16%)</td><td>Fischer Scientific</td><td>AA433689M</td><td>dilute to 4% before use</td></tr><tr><td>96-well plates</td><td>Greiner</td><td>655097</td><td>clear or black or clear bottom - black plates</td></tr><tr><td>Multichannel pipette (8 - 12 channels)</td><td></td><td></td><td></td></tr><tr><td>Reagent reservoirs</td><td></td><td></td><td></td></tr><tr><td>1x PBS</td><td></td><td></td><td></td></tr><tr><td>Microfuge tubes (0.6 ml)</td><td></td><td></td><td></td></tr><tr><td>Conicles (10 ml)</td><td></td><td></td><td></td></tr></tbody></table>

high throughput fluorometric technique for assessment of macrophage phagocytosis and actin polymerization

荧光分析的目的是作为一种有效的，成本有效的，高通量分析的吞噬作用和其他细胞过程的方法。这种技术可以用在各种细胞类型，包括粘附和非粘附的，以检查各种细胞特性。当研究吞噬作用，荧光技术利用吞噬细胞类型如巨噬细胞，和荧光标记的调理粒子的荧光可以以台盼蓝的存在而熄灭。下面粘附的巨噬细胞的电镀在96孔板中，荧光发色粒子（绿或红）施用和允许细胞吞噬对于不同量的时间。以下荧光体颗粒的内在化，细胞用台盼蓝，这有利于荧光信号的消光从其中不内化，或仅仅是附着于细胞表面的细菌。继锥虫洗涤，将细胞用PBS洗涤，固定，一次用DAPI染色（蓝色核荧光标记），其用于标记细胞的细胞核。通过板的核（蓝）读取或颗粒的简单荧光定量（红/绿）荧光我们可以检查的绿色相对荧光单位的比例:蓝色和确定一个表示每个细胞内在荧光的细菌量的吞噬指数。测定使用96孔的方法和多道移液器用于洗涤的持续时间，从吞噬数据采集结束的结束，是小于45分钟。流式细胞术以类似的方式也可以使用，但荧光的优点是它的高通量，评价与样品最小操纵和每个细胞的荧光强度的快速定量快速方法。类似的策略可以应用到非粘附细胞，活标记的细菌，肌动蛋白聚合，并且基本上任何利用荧光过程。因此，荧光是一种有前途的方法，其成本低，高throughpUT能力的细胞过程的研究。

通过使用该协议的量化吞噬作用和随后的肌动蛋白聚合的两种主要方法是观察连续或同步吞噬作用。 显微镜分析（ 图1B）示出了荧光图像媲美什么荧光正在记录（也参见图1）。在图1B是红色荧光的肌动蛋白，绿色荧光268 FITC的标签HKOP E.大肠杆菌 ，核DAPI染色，三个人269的合并后的图像荧光团在一起。这个图像显示有效的吞噬作用，...

Watch this Scientific Journal Video about High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization at JoVE.com

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

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 ...

high-throughput-fluorometric-technique-for-assessment-macrophage

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

14.6K 次观看.  University of Minnesota. 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.

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

"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 ...

科研

免疫与感染

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 ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

单细胞荧光显微镜定量 Efferocytosis 的研究

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a pET-SUMO-EGFP vector (SUMO = small ubiquitin-like modifier) and expressing enhanced green fluorescence protein (EGFP) in Escherichia coli (BL21DE). EGFP-expressing E. coli is coincubated with macrophages for 1 h at 37 &#176;C; the negative control group is incubated on ice for the same amount of time. Then, the macrophages are ready for assessment. The advantages of this technique include its simple and straightforward steps, and phagocytosis can be measured by both flow cytometer and fluorescence microscope. The EGFP-expressing E. coli are stable and display a strong fluorescence signal even after the macrophages are fixed with paraformaldehyde. This method is not only suitable for the assessment of macrophage cell lines or primary macrophages in vitro but also suitable for the evaluation of granulocyte and monocyte phagocytosis in peripheral blood mononuclear cells. The results show that the phagocytic capability of peritoneal macrophages from young (eight-week-old) mice is higher than that of macrophages from aged (16-month-old) mice. In summary, this method measures macrophage phagocytosis and is suitable for studying the innate immune system function.

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.

增强型绿色荧光蛋白表达大肠杆菌评价小鼠腹腔巨噬细胞吞噬细胞增多

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a ...

Mesenchymal Stem Cell Regulation of Macrophage Phagocytosis; Quantitation and Imaging

Mesenchymal stem cells (MSC) have traditionally been studied for their regenerative properties, but more recently, their immunoregulatory characteristics have been at the forefront. They interact with and regulate immune cell activity. The focus of this study is the MSC regulation of macrophage phagocytic activity. Macrophage (M&#934;) phagocytosis is an important part of the innate immune system response to infection, and the mechanisms through which MSC modulate this response are under active investigation. Presented here is a method to study M&#934; phagocytosis of non-opsonized zymosan particles conjugated to a pH-sensitive fluorescent molecule while in co-culture with MSC. As phagocytic activity increases and the labeled zymosan particles are enclosed within the acidic environment of the phagolysosome, the fluorescence intensity of the pH-sensitive molecule increases. With the appropriate excitation and emission wavelengths, phagocytic activity is measured using a fluorescent spectrophotometer and kinetic data is presented as changes in relative fluorescent units over a 70 min period. To support this quantitative data, the change in the phagocytic activity is visualized using dynamic imaging. Results using this method demonstrate that when in co-culture, MSC enhance M&#934; phagocytosis of non-opsonized zymosan of both naive and IFN-&#947; treated M&#934;. These data add to the current knowledge of MSC regulation of the innate immune system. This method can be applied in future investigations to fully delineate the underlying cellular and molecular mechanisms.

Presented here is a protocol to quantify and produce dynamic images of mesenchymal stem cell (MSC) mediated regulation of macrophage (M&#934;) phagocytosis of non-opsonized yeast (zymosan) particles that are conjugated to a pH-sensitive fluorescent molecule.

间充质干细胞调节巨噬细胞吞噬作用;定量和成像

Mesenchymal stem cells (MSC) have traditionally been studied for their regenerative properties, but more recently, their immunoregulatory characteristics ...

Flow Cytometry Approach to Characterize Phagocytic Properties of Acutely-Isolated Adult Microglia and Brain Macrophages In Vitro

Microglia and central nervous system (CNS)-infiltrating macrophages, collectively called CNS mononuclear phagocytes (CNS-MPs), play central roles in neurological diseases including neurodegeneration and stroke. CNS-MPs are involved in phagocytic clearance of pathological proteins, debris and neuronal synapses, each with distinct underlying molecular pathways. Characterizing these phagocytic properties can provide a functional readout that compliments molecular profiling of microglia using traditional flow cytometry, transcriptomics and proteomics approaches. Phagocytic profiling of microglia has relied on microscopic visualization and in vitro cultures of mouse neonatal microglia. The former approach suffers from limited sampling while the latter approach is inherently poorly reflective of the true in vivo state of adult CNS-MPs. This paper describes optimized protocols to phenotype phagocytic properties of acutely-isolated mouse CNS-MPs by flow cytometry. CNS-MPs are acutely isolated from adult mouse brain using mechanical dissociation followed by density gradient centrifugation, incubated with fluorescent microspheres or fluorescent A&#946; fibrils, washed, and then labeled with panels of antibodies against surface markers (CD11b, CD45). Using this approach, it is possible to compare phagocytic properties of brain-resident microglia with CNS-infiltrating macrophages and then assess the effect of aging and disease pathology on these phagocytic phenotypes. This rapid method also holds potential to functionally phenotype acutely-isolated human CNS-MPs from post-mortem or surgical brain specimens. Additionally, specific mechanisms of phagocytosis by CNS-MP subsets can be investigated by inhibiting select phagocytic pathways.

Assessment of phagocytic properties of microglia and brain macrophages can provide a valuable functional dimension to molecular profiling studies. We describe a validated protocol that uses flow cytometry to rapidly and reliably quantify phagocytosis of fluorescent microspheres and amyloid beta fibrils by acutely-isolated microglia and brain macrophages from mouse models.

Microglia and central nervous system (CNS)-infiltrating macrophages, collectively called CNS mononuclear phagocytes (CNS-MPs), play central roles in ...

神经科学

In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. Microglia clear up dead and dying neurons through the process of efferocytosis, a specialized form of phagocytosis. The phagocytosis function can be disrupted by environmental or genetic risk factors that affect microglia. This paper presents a rapid and simple in vitro&#160;microscopy protocol for studying microglial efferocytosis in an induced pluripotent stem cell (iPSC) model of microglia, using a human neuroblastoma cell line (SH-SY5Y) labeled with a pH-sensitive dye for the phagocytic cargo. The procedure results in a high yield of dead neuroblastoma cells, which display surface phosphatidylserine, recognized as an &#34;eat-me&#34; signal by phagocytes. The 96-well plate assay is suitable for live-cell time-lapse imaging, or the plate can be successfully fixed prior to further processing and quantified by high-content microscopy. Fixed-cell high-content microscopy enables the assay to be scaled up for screening of small molecule inhibitors or assessing the phagocytic function of genetic variant iPSC lines. While this assay was developed to study phagocytosis of whole dead neuroblastoma cells by iPSC-macrophages, the assay can be easily adapted for other cargoes relevant to neurodegenerative diseases, such as synaptosomes and myelin, and other phagocytic cell types.

Neurodegenerative diseases are associated with dysregulated microglia functions. This article outlines an in vitro assay of phagocytosis of neuroblastoma cells by iPSC-macrophages. Quantitative microscopy readouts are described for both live-cell time-lapse imaging and fixed-cell high-content imaging.

iPSC-巨噬细胞死亡神经母细胞的体外定量成像检测

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. ...

A Time-Efficient Fluorescence Spectroscopy-Based Assay for Evaluating Actin Polymerization Status in Rodent and Human Brain Tissues

Actin, the major component of cytoskeleton, plays a critical role in the maintenance of neuronal structure and function. Under physiological states, actin occurs in equilibrium in its two forms: monomeric globular (G-actin) and polymerized filamentous (F- actin). At the synaptic terminals, actin cytoskeleton forms the basis for critical pre- and post-synaptic functions. Moreover, dynamic changes in the actin polymerization status (interconversion between globular and filamentous forms of actin) are closely linked to plasticity-related alterations in synaptic structure and function. We report here a modified fluorescence-based methodology to assess polymerization status of actin in ex vivo conditions. The assay employs fluorescently labelled phalloidin, a phallotoxin that specifically binds to actin filaments (F-actin), providing a direct measure of polymerized filamentous actin. As a proof of principle, we provide evidence for the suitability of the assay both in rodent and post-mortem human brain tissue homogenates. Using latrunculin A (a drug that depolymerizes actin filaments), we confirm the utility of the assay in monitoring alterations in F-actin levels. Further, we extend the assay to biochemical fractions of isolated synaptic terminals wherein we confirm increased actin polymerization upon stimulation by depolarization with high extracellular K+.

We report a simple, time-efficient and high-throughput fluorescence spectroscopy-based assay for the quantification of actin filaments in ex vivo biological samples from brain tissues of rodents and human subjects.

用于评估啮齿动物和人类脑组织中行为素聚合状态的基于时间的荧光光谱分析

Actin, the major component of cytoskeleton, plays a critical role in the maintenance of neuronal structure and function. Under physiological states, actin ...

Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how fluorescent recovery after photobleaching (FRAP) and photoactivation techniques can be used to study the spatiotemporal dynamics of proteins in subcellular locations. We also show how these techniques enable straightforward determination of various parameters linked to actin cytoskeletal regulation and cell motility. Moreover, the microinjection of cells is additionally described as an alternative treatment (potentially preceding or complementing the aforementioned photomanipulation techniques) to trigger instantaneous effects of translocated proteins on cell morphology and function. Micromanipulation such as protein injection or local application of plasma membrane-permeable drugs or cytoskeletal inhibitors can serve as powerful tool to record immediate consequences of a given treatment on cell behavior at the single cell and subcellular level. This is exemplified here by immediate induction of lamellipodial cell edge protrusion by the injection of recombinant Rac1 protein, as established a quarter-century ago. In addition, we provide a protocol for determining the turnover of enhanced green fluorescent protein (EGFP)-VASP, an actin filament polymerase prominently accumulating at lamellipodial tips of B16-F1 cells, employing FRAP and including associated data analysis and curve fitting. We also present guidelines for estimating the rates of lamellipodial actin network polymerization, as exemplified by cells expressing EGFP-tagged &#946;-actin. Finally, instructions are given for how to investigate the rates of actin monomer mobility within the cell cytoplasm, followed by actin incorporation at sites of rapid filament assembly, such as the tips of protruding lamellipodia, using photoactivation approaches. None of these protocols is&#160;restricted to components or regulators of the actin cytoskeleton, but can easily be extended to explore in analogous fashion the spatiotemporal dynamics and function of proteins in various different subcellular structures or functional contexts.

We describe how micro- and photomanipulation techniques such as FRAP and photoactivation enable the determination of motility parameters and the spatiotemporal dynamics of proteins within migrating cells. Experimental readouts include subcellular dynamics and turnover of motility regulators or of the underlying actin cytoskeleton.

微操作系统技术允许分析骨架调节器的形态发生动力学和营业额

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how ...

生物学

Automated Imaging and Analysis for the Quantification of Fluorescently Labeled Macropinosomes

Macropinocytosis is a non-specific fluid-phase uptake pathway that allows cells to internalize large extracellular cargo, such as proteins, pathogens, and cell debris, through bulk endocytosis. This pathway plays an essential role in a variety of cellular processes, including the regulation of immune responses and cancer cell metabolism. Given this importance in biological function, examining cell culture conditions can provide valuable information by identifying regulators of this pathway and optimizing conditions to be employed in the discovery of novel therapeutic approaches. The study describes an automated imaging and analysis technique using standard laboratory equipment and a cell imaging multi-mode plate reader for the rapid quantification of the macropinocytic index in adherent cells. The automated method is based on the uptake of high molecular weight fluorescent dextran and can be applied to 96-well microplates to facilitate assessments of multiple conditions in one experiment or fixed samples mounted onto glass coverslips. This approach is aimed at maximizing reproducibility and reducing experimental variation while being both time-saving and cost-effective.

Automated assays using multi-well microplates are advantageous approaches for identifying pathway regulators by allowing the assessment of a multitude of conditions in a single experiment. Here, we have adapted the well-established macropinosome imaging and quantification protocol to a 96-well microplate format and provide a comprehensive outline for automation using a multi-mode plate reader.

用于荧光标记大促卵体定量的自动成像和分析

Macropinocytosis is a non-specific fluid-phase uptake pathway that allows cells to internalize large extracellular cargo, such as proteins, pathogens, and ...