In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro

The in vitro scratch assay is an easy, low-cost and well-developed method to measure cell migration in vitro. The basic steps involve creating a "scratch" in a cell monolayer, capturing the images at the beginning and at regular intervals during cell migration to close the scratch, and comparing the images to quantify the migration rate of the cells. Compared to other methods, the in vitro scratch assay is particularly suitable for studies on the effects of cell-matrix and cell-cell interactions on cell migration, mimic cell migration during wound healing in vivo and are compatible with imaging of live cells during migration to monitor intracellular events if desired. Besides monitoring migration of homogenous cell populations, this method has also been adopted to measure migration of individual cells in the leading edge of the scratch. Not taking into account the time for transfection of cells, in vitro scratch assay per se usually takes from several hours to overnight.     

Using the scratch assay to study cell migration in an inquiry-based cell biology lab

Cell migration, a fundamental process in development, wound healing, and immune function, is a common topic in undergraduate cell biology courses. We developed laboratory exercises with an inquiry-based learning (IBL) approach in which cell migration could be examined with the scratch assay, adapted from the primary literature. A narrow scratch was created in a confluent monolayer of cells growing on the bottom of a cell culture dish. Migration into the resultant cell-free zone from both sides of the scratch was measured after one day using the scale bar function of a digital camera. The Chinese hamster ovary cell line was used, but any adherent cell type could be examined. Students used the scratch assay to formulate hypotheses and design experiments in which variables affecting cell migration could be investigated. For example, the effect of cytoskeletal disruption was evaluated by adding the microtubule- and microfilament-disrupting drugs, colcemid and phallacidin, respectively, to the growth medium when the scratch was made. Optimal drug concentration parameters were determined for students to reference. Low drug concentrations inhibited cell migration, while higher concentrations killed the cells. This study demonstrated that the scratch assay is an accessible IBL method for studying cell migration.     

Three-dimensional traction force microscopy: a new tool for quantifying cell-matrix interactions

The interactions between biochemical processes and mechanical signaling play important roles during various cellular processes such as wound healing, embryogenesis, metastasis, and cell migration. While traditional traction force measurements have provided quantitative information about cell matrix interactions in two dimensions, recent studies have shown significant differences in the behavior and morphology of cells when placed in three-dimensional environments. Hence new quantitative experimental techniques are needed to accurately determine cell traction forces in three dimensions. Recently, two approaches both based on laser scanning confocal microscopy have emerged to address this need. This study highlights the details, implementation and advantages of such a three-dimensional imaging methodology with the capability to compute cellular traction forces dynamically during cell migration and locomotion. An application of this newly developed three-dimensional traction force microscopy (3D TFM) technique to single cell migration studies of 3T3 fibroblasts is presented to show that this methodology offers a new quantitative vantage point to investigate the three-dimensional nature of cell-ECM interactions.     

A quantitative, facile, and high-throughput image-based cell migration method is a robust alternative to the scratch assay

Cell migration is a key phenotype for a number of therapeutically important biological responses, including angiogenesis. A commonly used method to assess cell migration is the scratch assay, which measures the movement of cells into a wound made by physically scoring a confluent cell monolayer to create an area devoid of cells. Although this method has been adequate for qualitative characterization of migration inhibitors, it does not provide the highly reproducible results required for quantitative compound structure-activity relationship evaluation because of the inconsistent size and placement of the wound area within the microplate well. The Oris? Cell Migration Assay presents a superior alternative to the scratch assay, permitting formation of precisely placed and homogeneously sized cell-free areas into which migration can occur without releasing factors from wounded or dead cells or damaging the underlying extracellular matrix. Herein the authors compare results from the scratch and Oris? cell migration assays using an endothelial progenitor cell line and the Src kinase inhibitor dasatinib. They find that using the Acumen? Explorer laser microplate cytometer in combination with the Oris? Cell Migration Assay plate provides a robust, efficient, and cost-effective cell migration assay exhibiting excellent signal to noise, plate uniformity, and statistical validation metrics.     

非折叠蛋白质应答对人胚肾细胞293A迁移特性的影响

为了研究非折叠蛋白质应答对器官发生的影响,应用衣霉素诱导非折叠蛋白质应答并观察其对人胚肾细胞系293A迁移特性的影响。在实验中,应用划痕法对细胞迁移进行观察,并应用细胞黏附实验、荧光染色技术、扫描电镜技术及免疫印迹实验分别对细胞黏附特性、微管及微丝、细胞表面边缘的突起及小分子GTPase的表达水平进行研究。结果表明,非折叠蛋白质应答可以抑制细胞迁移,进一步的研究发现,非折叠蛋白质应答可以降低细胞的黏附能力、引起细胞骨架的重排、抑制伪足的形成并降低RhoA的表达水平。这提示,非折叠蛋白质应答可能通过抑制应激细胞的迁移为应激细胞的功能修复赢得了时间,在器官发生过程中发挥作用。     

Electrical fields in wound healing—An overriding signal that directs cell migration

Injury that disrupts an epithelial layer instantaneously generates endogenous electric fields (EFs), which were detected at human skin wounds over 150 years ago. Recent researches combining molecular, genetic and imaging techniques have provided significant insights into cellular and molecular responses to this “unconventional” signal. One unexpected finding is that the EFs play an overriding guidance role in directing cell migration in epithelial wound healing. In experimental models where other directional cues (e.g., contact inhibition release, population pressure etc.) are present, electric fields of physiological strength override them and direct cell migration. The electrotaxis or galvanotaxis is mediated by polarized activation of multiple signaling pathways that include PI3 kinases/Pten, membrane growth factor receptors and integrins. Genetic manipulation of PI3 kinase/Pten (Phosphoinositide 3-kinases/phosphatase and tensin homolog) and integrin β4 demonstrated the importance of those molecules. The electric fields are therefore a fundamental signal that directs cell migration in wound healing. One of the most challenging question is: How do cells sense the very weak electric signals? Clinically, it is highly desirable to develop practical and reliable technologies for wound healing management exploiting the electric signaling.     

Heparin/heparan sulfate/CD44-v3 enhances cell migration in term placenta-derived immortalized human trophoblast cells

The function of CD44-v3 and heparin/heparan sulfate (HS) signaling was investigated during trophoblast cell migration to identify their role in the renewal of syncytial layer damage caused by increased hemodynamic turbulence in the intervillous space and maintenance of syncytial integrity in pre-eclampsia. We evaluated the effect of heparin/HS/CD44-v3-mediated processes during scratch wound closure in monolayer immortalized human trophoblast cells derived from term placenta (TCL-1 cells). Western blot analysis showed that these cultured human trophoblast cells express the epidermal growth factor receptor and CD44-v3 but do not express syndecan 4. An in vitro scratch wound healing assay showed enhanced migration of trophoblast cells in a dose-dependent manner in the presence of heparin compared with controls when cultured under serum-free conditions. Conversely, an anti-CD44 function-blocking antibody and CD44 siRNA suppressed the migration of trophoblast cells in the presence of heparin in a similar scratch assay. Furthermore, both heparin treatment and in vitro scratch wounding induced the phosphorylation of p21-activated kinase 1 (PAK1), whereas the anti-CD44-v3 antibody suppressed the heparin-induced phosphorylation of PAK1 in trophoblast cells. These results indicate that heparin/HS/CD44-v3-mediated signaling, in the absence of growth factor networks, enhances the direct repair of the damaged trophoblast layer through the migration of trophoblast cells. This renewed cell coverage may lead to the maintenance of syncytiotrophoblast cell function and an associated reduction in pathogenic soluble factors derived from the damaged trophoblast cells.     

Localized elasticity measured in epithelial cells migrating at a wound edge using atomic force microscopy

Restoration of lung homeostasis following injury requires efficient wound healing by the epithelium. The mechanisms of lung epithelial wound healing include cell spreading and migration into the wounded area and later cell proliferation. We hypothesized that mechanical properties of cells vary near the wound edge, and this may provide cues to direct cell migration. To investigate this hypothesis, we measured variations in the stiffness of migrating human bronchial epithelial cells (16HBE cells) approximately 2 h after applying a scratch wound. We used atomic force microscopy (AFM) in contact mode to measure the cell stiffness in 1.5-microm square regions at different locations relative to the wound edge. In regions far from the wound edge (>2.75 mm), there was substantial variation in the elastic modulus in specific cellular regions, but the median values measured from multiple fields were consistently lower than 5 kPa. At the wound edge, cell stiffness was significantly lower within the first 5 microm but increased significantly between 10 and 15 microm before decreasing again below the median values away from the wound edge. When cells were infected with an adenovirus expressing a dominant negative form of RhoA, cell stiffness was significantly decreased compared with cells infected with a control adenovirus. In addition, expression of dominant negative RhoA abrogated the peak increase in stiffness near the wound edge. These results suggest that cells near the wound edge undergo localized changes in cellular stiffness that may provide signals for cell spreading and migration.     

The cell biology of suturing tendons

Trauma by suturing tendon form areas devoid of cells termed “acellular zones” in the matrix. This study aimed to characterise the cellular insult of suturing and acellular zone formation in mouse tendon. Acellular zone formation was evaluated using single grasping sutures placed using flexor tendons with time lapse cell viability imaging for a period of 12 h. Both tension and injury were required to induce cell death and cell movement in the formation of the acellular zone. DNA fragmentation studies and transmission electron microscopy indicated that cells necrosed.Parallel in vivo studies showed that cell-to-cell contacts were disrupted following grasping by the suture in tensioned tendon. Without tension, cell death was lessened and cell-to-cell contacts remained intact. Quantitative immunohistochemistry and 3D cellular profile mapping of wound healing markers over a one year time course showed that acellular zones arise rapidly and showed no evidence of healing whilst the wound healing response occurred in the surrounding tissues. The acellular zones were also evident in a standard modified “Kessler” clinical repair. In conclusion, the suture repair of injured tendons produces acellular zones, which may potentially cause early tendon failure.     

Hybrid liposomes inhibit growth and invasion of human osteosarcoma cells leading to apoptosis

Marked inhibitory effects of hybrid liposomes (HL-n; n=21, 23, 25) composed of 90 mol% l-α-dimyristoylphosphatidylcholine (DMPC) and 10 mol% polyoxyethylene(n) dodecyl ethers on the growth of two human osteosarcoma cell lines (MG-63 and U-2 OS) were obtained. Furthermore, fluorescence microscopic and flow cytometric analyses revealed the induction of apoptosis by HL-n in both cells. It is noteworthy that HL-23 could inhibit the invasion and migration of U-2 OS cells on the basis of matrigel invasion assay and scratch wound assay, respectively.     

Crucial role of vinexin for keratinocyte migration in vitro and epidermal wound healing in vivo

In the process of tissue injury and repair, epithelial cells rapidly migrate and form epithelial sheets. Vinexin is a cytoplasmic molecule of the integrin-containing cell adhesion complex localized at focal contacts in vitro. Here, we investigated the roles of vinexin in keratinocyte migration in vitro and wound healing in vivo. Vinexin knockdown using siRNA delayed migration of both HaCaT human keratinocytes and A431 epidermoid carcinoma cells in scratch assay but did not affect cell proliferation. Induction of cell migration by scratching the confluent monolayer culture of these cells activated both EGFR and ERK, and their inhibitors AG1478 and U0126 substantially suppressed scratch-induced keratinocyte migration. Vinexin knockdown in these cells inhibited the scratch-induced activation of EGFR, but not that of ERK, suggesting that vinexin promotes cell migration via activation of EGFR. We further generated vinexin (−/−) mice and isolated their keratinocytes. They similarly showed slow migration in scratch assay. Furthermore, vinexin (−/−) mice exhibited a delay in cutaneous wound healing in both the back skin and tail without affecting the proliferation of keratinocytes. Together, these results strongly suggest a crucial role of vinexin in keratinocyte migration in vitro and cutaneous wound healing in vivo.     

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.     

S100A4 downregulates filopodia formation through increased dynamic instability

Cell migration requires the initial formation of cell protrusions, lamellipodia and/or filopodia, the attachment of the leading lamella to extracellular cues and the formation and efficient recycling of focal contacts at the leading edge. The small calcium binding EF-hand protein S100A4 has been shown to promote cell motility but the direct molecular mechanisms responsible remain to be elucidated. In this work, we provide new evidences indicating that elevated levels of S100A4 affect the stability of filopodia and prevent the maturation of focal complexes. Increasing the levels of S100A4 in a rat mammary benign tumor derived cell line results in acquired cellular migration on the wound healing scratch assay. At the cellular levels, we found that high levels of S100A4 induce the formation of many nascent filopodia, but that only a very small and limited number of those can stably adhere and mature, as opposed to control cells, which generate fewer protrusions but are able to maintain these into more mature projections. This observation was paralleled by the fact that S100A4 overexpressing cells were unable to establish stable focal adhesions. Using different truncated forms of the S100A4 proteins that are unable to bind to myosin IIA, our data suggests that this newly identified functions of S100A4 is myosin-dependent, providing new understanding on the regulatory functions of S100A4 on cellular migration.     

Downregulation of CD9 in Keratinocyte Contributes to Cell Migration via Upregulation of Matrix Metalloproteinase-9

Tetraspanin CD9 has been implicated in various cellular and physiological processes, including cell migration. In our previous study, we found that wound repair is delayed in CD9-null mice, suggesting that CD9 is critical for cutaneous wound healing. However, many cell types, including immune cells, endothelial cells, keratinocytes and fibroblasts undergo marked changes in gene expression and phenotype, leading to cell proliferation, migration and differentiation during wound repair, whether CD9 regulates kerationcytes migration directly remains unclear. In this study, we showed that the expression of CD9 was downregulated in migrating keratinocytes during wound repair in vivo and in vitro. Recombinant adenovirus vector for CD9 silencing or overexpressing was constructed and used to infect HaCaT cells. Using cell scratch wound assay and cell migration assay, we have also demonstrated that downregulation of CD9 promoted keratinocyte migration in vitro, whereas CD9 overexpression inhibited cell migration. Moreover, CD9 inversely regulated the activity and expression of MMP-9 in keratinocytes, which was involved in CD9-regulated keratinocyte migration. Importantly, CD9 silencing-activated JNK signaling was accompanied by the upregulation of MMP-9 activity and expression. Coincidentally, we found that SP600125, a JNK pathway inhibitor, decreased the activity and expression of MMP-9 of CD9-silenced HaCaT cells. Thus, our results suggest that CD9 is downregulated in migrating keratinocytes in vivo and in vitro, and a low level of CD9 promotes keratinocyte migration in vitro, in which the regulation of MMP-9 through the JNK pathway plays an important role.     

Live cell imaging: approaches for studying protein dynamics in living cells

In the last decade, the long-standing biologist's dream of seeing the molecular events within the living cell came true. This technological achievement is largely due to the development of fluorescence microscopy technologies and the advent of green fluorescent protein as a fluorescent probe. Such imaging technologies allowed us to determine the subcellular localization, mobility and transport pathways of specific proteins and even visualize protein-protein interactions of single molecules in living cells. Direct observation of such molecular dynamics can provide important information about cellular events that cannot be obtained by other methods. Thus, imaging of protein dynamics in living cells becomes an important tool for cell biology to study molecular and cellular functions. In this special issue of review articles, we review various imaging technologies of microscope hardware and fluorescent probes useful for cell biologists, with a focus on recent development of live cell imaging.     

Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and inflammatory responses. Given the current interest in the role of mesenchymal stromal cells in mediating tissue repair, we are interested in quantifying the migratory capacity of these cells, and understanding how migratory capacity may be altered after damage. Optimization of a rigorously quantitative migration assay that is both easy to customize and cost-effective to perform is key to answering questions concerning alterations in cell migration in response to various stimuli. Current methods for quantifying cell migration, including scratch assays, trans-well migration assays (Boyden chambers), micropillar arrays, and cell exclusion zone assays, possess a range of limitations in reproducibility, customizability, quantification, and cost-effectiveness. Despite its prominent use, the scratch assay is confounded by issues with reproducibility related to damage of the cell microenvironment, impediments to cell migration, influence of neighboring senescent cells, and cell proliferation, as well as lack of rigorous quantification. The optimized scratch assay described here demonstrates robust outcomes, quantifiable and image-based analysis capabilities, cost-effectiveness, and adaptability to other applications.     

去甲斑蝥素对H446 细胞Id1mRNA 表达的影响

目的:观察去甲斑蝥素对小细胞肺癌H446细胞Id1mRNA表达的影响。方法:分别利用MTT法检测细胞生长活性;用划痕实验分析细胞迁移能力;采用Hoechst染色观察细胞凋亡;用实时荧光RT-PCR法测定H446中Id1mRNA的表达。结果:去甲斑蝥素对H446细胞的生长有明显的抑制作用,细胞的生长抑制率和凋亡率明显增加,细胞迁移距离明显缩短。去甲斑蝥素可抑制细胞内Id1mRNA的表达,其相对定量随去甲斑蝥素的浓度增大而减少。结论:在H446细胞中,去甲斑蝥素能抑制Id1mRNA的表达,这可能是去甲斑蝥素抑制细胞生长,迁移和诱导细胞凋亡的重要机制之一。