PhagoKinetic Track Assay: Imaging and Analysis of Single Cell Migration
吞噬动力追踪试验：单细胞迁移的成像和分析
发布: 2016年01月05日第6卷第1期 DOI: 10.21769/BioProtoc.1699 浏览次数: 10252
评审: Guillaume Bompard Shannon RuppertAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Apr 2015
Abstract
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.
Microscopy (显微镜)
High throughput (高吞吐量)
Screening (筛选)
Image analysis (图像分析)
Materials and Reagents
- 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 ),
- MDA-MB-417.5 (kindly provided by Joan Massagué) and Hs578T (ATCC, catalog number: HBT-126 )]
- Phosphate buffered saline (PBS) without Ca/Mg (any supplier)
- Trypsin-EDTA (Life Technologies, InvitrogenTM)
- Fibronectin from bovine plasma (Sigma-Aldrich, catalog number: F1141 )
- 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 )
- Black 96-well flat-bottom microscopy plates (µClear 96-well plate) (Greiner Bio-One GmbH, catalog number: 655090 )
- 4% Formaldehyde (any supplier) in PBS
- RPMI medium (Life Technologies)
- Fetal Bovine Serum (FBS) (PAA Laboratories)
- Penicillin-Streptomycin (Penicillin-Streptomycin) (Life Technologies, Gibco®)
- Complete medium (see Recipes)
Equipment
- Cell culture set up, including laminar flow hood, cell culture incubator (37 °C and 5% CO2), cell counter or counting chamber, multichannel pipets
- 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
- Hydroflex plate washer (Tecan Trading AG)
Software
- WIS-PhagoTracker v2_3_12 (Weizmann Institute of Science, http://www.weizmann.ac.il/vet/IC/software/wis-phagotracker; open source software)
- Montage2MRC (Weizmann Institute of Science, http://www.weizmann.ac.il/vet/IC/software/wis-phagotracker; open source software)
- KNIME (optional) (www.knime.org; open source software)
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Fokkelman, M., Roosmalen, W. V., Rogkoti, V., Le Dévédec, S. E., Geiger, B. and Water, B. V. D. (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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分类
癌症生物学 > 通用技术 > 细胞生物学试验
癌症生物学 > 侵袭和转移 > 细胞生物学试验
细胞生物学 > 细胞成像 > 活细胞成像
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