PhagoKinetic Track Assay: Imaging and Analysis of Single Cell Migration

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Cell migration is a highly complex and dynamic biological process, essential in several physiological phenomena and pathologies including cancer dissemination and metastasis formation. Thus understanding single cell migration is highly relevant and requires a suitable image-based assay. Depending on the speed of the moving cells, one may require fast time-lapse microscopy, which is not always suitable for high-throughput screening. To overcome this, a quantitative and fixed single cell migration assay was developed based on the PhagoKinetic Tracks (PKT) procedure. Briefly, cells are seeded on top of a monolayer of carboxylated latex beads, and as cells migrate, they phagocytose these beads and leave behind a migratory track. These bead-free migratory tracks can be visualized using a standard bright field microscope and analysed for a multiparametric quantitative assessment of single cell migration (Naffar-Abu-Amara et al., 2008).
Here we describe a detailed and optimized protocol of the PKT assay, adaptable for both RNAi and drug screening (van Roosmalen et al., 2015). This protocol allows the user to study migratory behaviour at the single cell level, without fast and live-imaging microscopy.

Keywords: Cell migration(细胞迁移), Microscopy(显微镜), High throughput(高吞吐量), Screening(筛选), Image analysis(图像分析)

Materials and Reagents

  1. Tumour cells [in particular, this assay was set up for H1299 non-small cell lung carcinoma cells (ATCC, catalog number: CRL-5803 ) and breast cancer cell lines MDA-MB-231 (ATCC, catalog number: HBT-26 ),
  2. MDA-MB-417.5 (kindly provided by Joan Massagué) and Hs578T (ATCC, catalog number: HBT-126 )]
  3. Phosphate buffered saline (PBS) without Ca/Mg (any supplier)
  4. Trypsin-EDTA (Life Technologies, InvitrogenTM)
  5. Fibronectin from bovine plasma (Sigma-Aldrich, catalog number: F1141 )
  6. White carboxylate-modified latex beads (CML Latex Beads, 4% w/v, 0.4 µm), and number of particles is given in the MSDS/COA documents provided by the manufacturer (Thermo Fisher Scientific, catalog number: C37238 )
  7. Black 96-well flat-bottom microscopy plates (µClear 96-well plate) (Greiner Bio-One GmbH, catalog number: 655090 )
  8. 4% Formaldehyde (any supplier) in PBS
  9. RPMI medium (Life Technologies)
  10. Fetal Bovine Serum (FBS) (PAA Laboratories)
  11. Penicillin-Streptomycin (Penicillin-Streptomycin) (Life Technologies, Gibco®)
  12. Complete medium (see Recipes)


  1. Cell culture set up, including laminar flow hood, cell culture incubator (37 °C and 5% CO2), cell counter or counting chamber, multichannel pipets
  2. BD Pathway 855 BioImager (BD Biosciences); or another brand or type of inverted microscope equipped with brightfield illumination, automated stage, 10x objective and software for automated imaging
  3. Hydroflex plate washer (Tecan Trading AG)


  1. WIS-PhagoTracker v2_3_12 (Weizmann Institute of Science, http://www.weizmann.ac.il/vet/IC/software/wis-phagotracker; open source software)
  2. Montage2MRC (Weizmann Institute of Science, http://www.weizmann.ac.il/vet/IC/software/wis-phagotracker; open source software)
  3. KNIME (optional) (www.knime.org; open source software)


  1. Day 1
    1. In a sterile laminar flow hood, add 40 µl/well fibronectin (10 µg/ml in PBS) to a 96-well µClear plate. Transfer the plate to a cell culture incubator and incubate for 1 h at 37 °C.
    2. Aspirate fibronectin without scratching the surface, and wash twice with PBS (preferentially using plate washer). Add 100 µl PBS per well and proceed to the next step.
    3. Swirl the stock flask of CML latex beads and aliquot 250 µl beads (approximately 3.25 x 1011 particles) per plate and spin down at 13,000 rpm for 5 min. Discard supernatant and resuspend bead pellet in 1 ml PBS. Vortex for 1 min and spin down at 13,000 rpm for 5 min. Remove supernatant and resuspend bead pellet in 1 ml PBS. Transfer resuspended beads to 15 or 50 ml tube and add PBS to a final beads suspension volume of 7 ml per plate.
    4. Remove PBS from fibronectin-coated plate without scratching the surface and add 70 µl beads per well.
    5. Visually check the plate and make sure the surface of the well is completely covered with beads and incubate for 1 h at 37 °C.
    6. Use plate-washer and gently wash 7 times with PBS (200 µl per well). If no plate-washer is available, carefully wash the plate manually using a multichannel and ensure the bead-coated surface is not scratched. After washing, add 100 µl PBS per well.
    7. Wrap the plate in parafilm and store plate at 4 °C for next day use.
    8. We would advise to prepare fresh beads for each experiment (as explained above) and avoid storage for long period.

  2. Day 2
    1. Carefully aspirate PBS from bead-coated plate and replace with 100 µl complete medium.
    2. Prepare cells (e.g., from culture flask, or after siRNA transfection) and resuspend into single cell suspension.
    3. Seed 50-200 cells in a 50 µl single cell suspension (depending on cell size & migration capacity) and return plate to cell culture incubator. The final volume is 150 μl (100 µl complete medium + 50 µl single cell suspension) in which 50-200 cells will be allowed to form single-cell migration tracks.
    4. We recommend that cells are allowed to attach for 1 h before adding chemical compounds or inhibitors.
    5. Depending on migration capacity, fixate cells after 7-12 h of cell migration by removing 140 µl medium from the plate and adding 70 µl 4% formaldehyde (final formaldehyde concentration is 3.5%). Fixate for 10 min at RT.
    6. Wash plate twice with PBS (200 µl per well) without scratching the bead-coated surface.
    7. Wrap the plate in parafilm and store at 4 °C until imaging.

      Figure 1. Microscopy set up of PhagoKinetic track assay. A. Non-homogenous illumination in the original brightfield image is minimized by adding a light diffuser (tip box lid), providing a clear image with equal intensities. B. A seamless montage of the largest square without cell borders (red box) allows the user to record a maximal number of complete cell tracks.

  3. Imaging and analysis
    1. Use a brightfield microscope with 10x objective to visualize bead-free migratory tracks. Whole-well montages were obtained as followed:
      1. The transmitted light source is above the 96-plate. For equal illumination, place a tipbox lid on top of the plate to diffuse the light (see Figure 1a).
      2. Use laser autofocus in each well to focus before acquisition.
      3. Use montage-settings to acquire the largest montage of the well without imaging well-borders (example in Figure 1b).
    2. Use Montage2MRC to convert all montages of one plate into a single r6d-file, following the software instructions.
    3. Run PhagoTracker software and analyse images according to the software guidelines (Figure 2a-b). The PhagoTracker user guide is very detailed and contains a myriad of information to optimize your image analysis. Briefly, the following settings were used:
      1. In pre-processing, set Correct Illumination to ‘Non’.
      2. Use a High Pass & Low Pass filter, and set the Adjust Contrast to ‘Whole Well’.
      3. For Track Detection, we use the recommended threshold settings. Depending on gray values, overall intensity, cell size and track size, you might need to find optimal settings.
      4. Use Track Filtering to reject tracks without cells and to reject tracks with more than 1 cell. Briefly, the software detects bright objects (migratory paths) and dark objects (cells). It subsequently determines whether each migratory path is accepted (1 path with 1 cell) or rejected (1 path with 0 cells, or 1 path with >1 cells). Tracks can also be manually accepted or rejected after analysis. This is not necessary if the image analysis parameters are set correctly.
    4. PhagoTracker calculates multiple morphometric features for each migratory track, including Total Area, Net Area, Minor Axis, Major Axis, Axial Ratio and Perimeter as measurements of track size and shape. Furthermore, the Solidity and Roughness of each track are calculated, which represent membrane activity and ruffling during migration (Figure 2c-d). The software generates 2 files with quantitative output: one file contains data of all accepted individual tracks, the other contains average values and standard deviations of accepted, rejected and all tracks per well.
    5. Quantitative output can be further analysed using PhagoTracker (see user guide), or processed using preferred programs, like Excel, Graphpad, R or KNIME.
      1. To automate your analysis, KNIME is recommended. This open-source software contains a plethora of ready-to-use analysis nodes, including a plate lay out joiner, different normalization methods (Z-score, B-score, NPI, POC, etc.) and plotting tools (2D/3D scatter plot, etc.), to build an analysis pipeline.

        Figure 2. PhagoTracker software and image analysis procedure. A. The PhagoTracker software interface. B. PhagoTracker software performs image analysis in several steps. Images are pre-processed to enhance contrast and correct illumination. Multi-Scale segmentation finds candidate cell and path segments (shown in orange and blue, respectively) which are subsequently combined into track segments. The candidate tracks are filtered based on the selection criteria and are marked as confirmed (in red) or rejected (in blue). C. Representative images of Hs578T breast cancer cells. Control cells show large elongated tracks with a rough border. Cells with siRNA knockdown (KD) do not migrate and form round tracks. D. Quantification of individual tracks shown in C. Solidity is calculated as (track area/ area of the convex hull) and roughness as (perimeter2) / (4π*Total Area).


Important considerations

  1. Concerning the plate preparation:
    1. There is a very small, negligible, risk that you wash away the beads, but this only occurs if you use a lot of force. In reality, this problem never occurs.
    2. It is very easy to gently tap or shake (horizontally) the plate to distribute the beads solution and cover the surface of the wells. This protocol uses an excess of beads and beads are coated by sedimentation, so the coverage is very uniform.
  2. At all times, do not scratch the bottom of the plate. Scratches in the bead-coating can appear as migratory tracks and could affect your analysis. In general, minor/small scratches can be ignored, as they do not affect the analysis. Indeed, in case a scratch is detected as track, there is no cell in it and therefore it would be rejected.
  3. This protocol can be used for other cell lines, however we recommend a pilot experiment to find optimal conditions for any cell line used. These include:
    1. Size, type and surface charge of the beads. Different sizes, types and charge of beads have been tested before and are clearly described by Naffar-Abu-Amara et al. Briefly, CML latex beads with a size of 340 or 400 nm work best. Beads with a low charge density will interact strongly with the surface of the well and consequently cells cannot remove the beads as they migrate. The beads used in this protocol have a charge density of 85-100 µEq/g and can be used for a wide range of cells. The catalog number is provided in the materials and reagents section allowing users to order the same beads.
    2. Optimal cell density, which is dictated by several parameters such as cell size (the larger the cells, the lower the density) and cell motility (the higher the speed, the lower the cell density).
    3. Time of migration. Less motile cell lines might require longer incubation time for migratory track formation.
      Aim for a maximum number of bead-free migratory tracks at the end of the assay that do not intersect. Track intersection can be inspected visually. You see 1 bright track and 2 cells, indicating either intersection or cell division (Figure 2b, first panel). The PhagoTracker software saves the path (migratory tracks) and cells image that is generated during analysis (Figure 2b, third panel). You can decide whether tracks are automatically rejected or not, and which criteria to use for rejection.


  1. Complete medium
    RPMI medium was supplemented with 10% fetal bovine serum and 25 IU/ml penicillin and 25 µg/ml streptomycin.


This protocol was developed based on Naffar-Abu-Amara et al. (2008) and further optimized in the Division of Toxicology, Leiden Academic Centre for Drug Research, Leiden University. The work was funded by grants from the EU-FP7 - Systems Microscopy NoE (grant no. 258068 to B. van de Water) and the Dutch Cancer Society (UL2007-3860).


  1. Naffar-Abu-Amara, S., Shay, T., Galun, M., Cohen, N., Isakoff, S. J., Kam, Z. and Geiger, B. (2008). Identification of novel pro-migratory, cancer-associated genes using quantitative, microscopy-based screening. PLoS One 3(1): e1457.
  2. Sharon, E., Galun, M., Sharon, D., Basri, R. and Brandt, A. (2006). Hierarchy and adaptivity in segmenting visual scenes. Nature 442(7104): 810-813.
  3. van Roosmalen, W., Le Devedec, S. E., Golani, O., Smid, M., Pulyakhina, I., Timmermans, A. M., Look, M. P., Zi, D., Pont, C., de Graauw, M., Naffar-Abu-Amara, S., Kirsanova, C., Rustici, G., Hoen, P. A., Martens, J. W., Foekens, J. A., Geiger, B. and van de Water, B. (2015). Tumor cell migration screen identifies SRPK1 as breast cancer metastasis determinant. J Clin Invest 125(4): 1648-1664.


在这里,我们描述了PKT测定的详细和优化的方案,适用于RNAi和药物筛选(van Roosmalen等人,2015)。这个协议允许用户在单细胞水平研究迁移行为,没有快速和活成像显微镜。

关键字:细胞迁移, 显微镜, 高吞吐量, 筛选, 图像分析


  1. 肿瘤细胞[特别地,该测定用于H1299非小细胞肺癌细胞(ATCC,目录号:CRL-5803)和乳腺癌细胞系MDA-MB-231(ATCC,目录号:HBT-26) ,
  2. MDA-MB-417.5(由JoanMassagué友情提供)和Hs578T(ATCC,目录号:HBT-126)]
  3. 没有Ca/Mg(任何供应商)的磷酸盐缓冲盐水(PBS)
  4. 胰蛋白酶-EDTA(Life Technologies,Invitrogen )
  5. 来自牛血浆的纤连蛋白(Sigma-Aldrich,目录号:F1141)
  6. 在制造商(Thermo Fisher Scientific,目录号:C37238)提供的MSDS/COA文件中给出白色羧酸酯改性的乳胶珠(CML Latex Beads,4%w/v,0.4μm) >
  7. 黑色96孔平底显微镜板(μClear96孔板)(Greiner Bio-One GmbH,目录号:655090)
  8. 4%甲醛(任何供应商)在PBS
  9. RPMI培养基(Life Technologies)
  10. 胎牛血清(FBS)(PAA Laboratories)
  11. 青霉素 - 链霉素(青霉素 - 链霉素)(Life Technologies,Gibco )
  12. 完整媒介(见配方)


  1. 细胞培养装置,包括层流罩,细胞培养培养箱(37℃和5%CO 2),细胞计数器或计数室,多通道移液器
  2. BD Pathway 855 BioImager(BD Biosciences);或另一品牌或类型的倒置显微镜,配备明场照明,自动舞台,10x物镜和自动化成像软件
  3. Hydroflex洗板机(Tecan Trading AG)


  1. WIS-PhagoTracker v2_3_12(Weizmann Institute of Science, http://www.weizmann .ac.il/vet/IC/software/wis-phagotracker ;开源软件)
  2. Montage2MRC(Weizmann Institute of Science, http://www.weizmann.ac。 il/vet/IC/software/wis-phagotracker ;开源软件)
  3. KNIME(可选)( www.knime.org ;开源软件)


  1. 第1天
    1. 在无菌层流罩中,加入40μl/孔纤连蛋白(10μg/ PBS)至96孔μClear平板。将板转移到细胞培养物 培养箱中并在37℃下孵育1小时
    2. 吸出纤连蛋白 而不刮擦表面,并用PBS洗涤两次(优选 使用洗板机)。每孔加入100μlPBS,继续下一步 步骤。
    3. 旋转储备烧瓶的CML乳胶珠和等分试样250μl ?珠(约3.25×10 11个颗粒),并旋转 13,000rpm,5分钟。弃去上清液并重悬细胞小球在1 ?ml PBS。涡旋1分钟并在13,000rpm离心5分钟。去掉 上清液和重悬的珠粒在1ml PBS中。转移重悬 珠至15或50ml管,并加入PBS至最终珠悬浮液体积 ?每板7ml
    4. 从纤维连接蛋白包被板除去PBS,不刮表面,每孔添加70微升的珠。
    5. 目视检查板,确保井的表面 完全用珠子覆盖并在37℃下孵育1小时
    6. 使用 洗板,并用PBS(每孔200μl)轻轻洗涤7次。如果不 洗板机,可用手动小心清洗板 多通道,并确保珠涂层表面不被划伤。后 洗涤,每孔加入100μlPBS
    7. 包装在parafilm板和存储板在4°C第二天使用
    8. 我们建议为每个实验准备新鲜的珠子(如上所述),避免长期储存。

  2. 第2天
    1. 小心地从珠涂布板吸取PBS,并更换为100微升完全培养基
    2. 从培养瓶中制备细胞(例如,或siRNA转染后),并重悬于单细胞悬液中。
    3. 种子50-200细胞在50微升单细胞悬液(取决于 细胞大小&迁移能力)和返回板细胞培养 孵化器。最终体积为150μl(100μl完全培养基+50μl 单细胞悬浮液),其中将允许形成50-200个细胞 单细胞迁移轨迹。
    4. 我们建议允许细胞在加入化合物或抑制剂之前附着1小时
    5. 根据迁移能力,在细胞7-12小时后固定细胞 通过从板中移除140μl培养基并加入70μl4% 甲醛(最终甲醛浓度为3.5%)。固定为10 min
    6. 用PBS洗涤平板两次(每孔200μl),不划伤涂有珠子的表面
    7. 包装在parafilm板,存储在4°C,直到成像

      图1. PhagoKinetic轨迹测定的显微镜设置。。 原始明场图像中的非均匀照明是 通过添加光扩散器(提示盒盖)最小化,提供清晰 图像具有相等的强度。 B.最大的无缝蒙太奇 正方形没有单元格边框(红框)允许用户记录a 最大数量的完整单元轨道。

  3. 成像和分析
    1. 使用明场显微镜与10倍物镜可视化无珠 迁徙轨道。如下获得全井蒙太奇:
      1. 透射光源在96板的上方。为平等 照明,在平板的顶部放置小盒盖以扩散 光(见图1a)。
      2. 在获取之前,在每个孔中使用激光自动对焦进行对焦
      3. 使用蒙太奇设置获取井的最大蒙太奇,没有成像井边界(例如图1b)。
    2. 按照软件说明,使用Montage2MRC将一个版面的所有蒙太奇图像转换为单个r6d文件。
    3. 运行PhagoTracker软件并根据图像分析 软件指南(图2a-b)。 PhagoTracker用户指南非常 详细和包含大量的信息,以优化您的形象 分析。简单地说,使用以下设置:
      1. 在预处理中,将正确照明设置为"非"
      2. 使用高通&低通滤波器,并将"调整对比度"设置为"整体"
      3. 对于轨道检测,我们使用推荐的阈值设置。 根据灰度值,总强度,单元尺寸和轨道尺寸, 您可能需要找到最佳设置。
      4. 使用轨道过滤 拒绝没有单元的轨道和拒绝具有多于1个单元的轨道。 简而言之,软件检测明亮的物体(迁移路径)和黑暗 对象(单元格)。它随后确定每个洄游路径 (具有1个单元格的1个路径)或拒绝(1个路径,0个单元格,或1个 具有> 1个单元的路径)。轨道也可以手动接受或拒绝 ?分析后。如果图像分析参数不是必需的 设置正确。
    4. PhagoTracker计算多个形态测量 ?每个迁徙轨道的特征,包括总面积,净面积, 小轴,主轴,轴比和周长作为测量 轨道尺寸和形状。此外,每个的稠度和粗糙度 轨迹,其代表膜活性和波状起伏 (图2c-d)。软件生成2个文件 定量输出:一个文件包含所有接受的个人的数据 轨迹,另一个包含的平均值和标准偏差 接受,拒绝和每个井的所有轨道。
    5. 定量输出 可以使用PhagoTracker(请参阅用户指南)进行进一步分析 使用首选程序(如Excel,Graphpad,R或KNIME)进行处理。
      1. 为了自动化您的分析,建议使用KNIME。这个开源 软件包含大量即用型分析节点,包括a ?板铺板,不同的标准化方法(Z-score, B-score,NPI,POC,等)和绘图工具(2D/3D散点图,等) 以构建分析管道

        图2. PhagoTracker软件和 图像分析程序。 A. PhagoTracker软件界面。乙。 PhagoTracker软件在几个步骤中执行图像分析。图片 被预处理以增强对比度和正确的照明。 多尺度分割发现候选单元格和路径段(如图所示) 分别为橙色和蓝色),随后将其组合 轨道段。基于该选择来过滤候选轨迹 ?标准,并标记为确认(红色)或拒绝(蓝色)。 C。 ?Hs578T乳腺癌细胞的代表图像。对照细胞显示 ?大长条轨道与粗糙的边界。细胞用siRNA敲除 (KD)不迁移并形成圆形轨道。 D.量化 单个磁道,如C.中所示。密实度计算为(磁道面积/ 凸包的面积)和粗糙度为(周长<2/d>)/(4π*总 区)。



  1. 关于板的准备:
    1. 有一个非常小,可忽略的风险,你洗掉珠子, 但这只会发生,如果你使用很大的力。在现实中,这个问题 ?永远不会发生。
    2. 这是很容易轻轻拍打或摇晃 (水平)板以分布珠溶液并覆盖 表面的井。此协议使用过量的珠和珠 通过沉降涂覆,因此覆盖非常均匀。
  2. 在任何时候,不要划伤板的底部。珠涂层中的划痕可以表现为迁移轨迹,并可能影响分析。一般来说,小/小划痕可以忽略,因为它们不影响分析。事实上,如果划痕被检测为轨迹,则其中没有细胞,因此它将被拒绝
  3. 这个协议可以用于其他细胞系,但我们建议一个试点实验找到使用的任何细胞系的最佳条件。这些包括:
    1. 珠的尺寸,类型和表面电荷。不同尺寸,类型和 珠粒的电荷已经过测试,并且被清楚地描述 Naffar-Abu-Amara等人简要地说,大小为340的CML乳胶珠或 400 nm工作效果最好。具有低电荷密度的珠子将强烈相互作用 ?与孔的表面和因此细胞不能去除 珠子,因为他们迁移。在本协议中使用的珠子有电荷 密度为85-100μEq/g,可用于广泛的细胞。的 目录号在材料和试剂部分提供 允许用户订购相同的珠子。
    2. 最佳细胞密度, 这取决于几个参数,如单元格大小(更大 细胞,密度越低)和细胞运动性(越高 速度,细胞密度越低)。
    3. 迁移时间。较少的活动细胞系可能需要更长的迁移轨迹形成的孵育时间 瞄准最大数目的无珠迁移轨道在结束 该测定不相交。可以检查轨道交叉 视觉上。你看到1明亮的轨道和2个单元格,指示 交叉或细胞分裂(图2b,第一图)。 PhagoTracker ?软件保存路径(迁移轨迹)和细胞图像 (图2b,第三图)。你可以决定 是否自动拒绝磁道,以及哪些标准 用于拒绝。


  1. 完成媒介


该方案是基于Naffar-Abu-Amara等人(2008)开发的,并且在莱顿大学莱顿学术药物研究中心的毒理学部进一步优化。该工作由来自EU-FP7-系统显微镜NoE(批准号258068至B.van de Water)和荷兰癌症协会(UL2007-3860)的赠款资助。


  1. Naffar-Abu-Amara,S.,Shay,T.,Galun,M.,Cohen,N.,Isakoff,S.J.,Kam,Z.and Geiger,B。(2008)。 使用定量,基于显微镜的筛选鉴定新型促洄选,癌症相关基因。 a> PLoS One 3(1):e1457。
  2. Sharon,E.,Galun,M.,Sharon,D.,Basri,R。和Brandt,A。(2006)。 分割视觉场景中的层次和适应性 自然 442 (7104):810-813。
  3. van Roosmalen,W.,Le Devedec,SE,Golani,O.,Smid,M.,Pulyakhina,I.,Timmermans,AM,Look,MP,Zi,D.,Pont,C.,de Graauw, Naffar-Abu-Amara,S.,Kirsanova,C.,Rustici,G.,Hoen,PA,Martens,JW,Foekens,JA,Geiger,B.and van de Water, 肿瘤细胞迁移屏幕将SRPK1鉴定为乳腺癌转移决定因素。 Invest 125(4):1648-1664。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Fokkelman, M., Roosmalen, W. v., Rogkoti, V., Le Dévédec, S. E., Geiger, B. and Water, B. v. (2016). PhagoKinetic Track Assay: Imaging and Analysis of Single Cell Migration. Bio-protocol 6(1): e1699. DOI: 10.21769/BioProtoc.1699.

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