This approach is however demanding, tedious, and time-consuming. Even though not comparable with our study, similar results for wound closure were found by Walter et al. This is also the case with respect to our plugin where overestimations approached 6.5% 4.4 and 10.33% 6.9, respectively. Understanding the processes involved in this migration can provide potential targets for inhibition. Invest. 6, pp. Yes 1, pp. 30 0 obj
This value should always be greater than zero. successfully screened cytotoxic alkaloids for the ability to inhibit biological processes related to cell migration and cytoskeleton dynamics. Physiol., vol. (2014) [4]. ------------------------------------------------------. The variance window radius represents the radius of the variance filter, which we establish to determine the empty or the occupied zones. No, Is the Subject Area "Open source software" applicable to this article? 11 0 obj
It quantifies the area, wound coverage of total area, average wound width and width standard deviation. 41 0 obj
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Our results indicate that accurate analyses are likely to be found for variance window radii is between 3 to 25, threshold values in the range of 50 to 150 and percentages of saturated pixels between 0.001 to 0.4. 17, no. [14] T. Gebck, M. M. P. Schulz, P. Koumoutsakos, and M. Detmar, TScratch: a novel and simple software tool for automated analysis of monolayer wound healing assays: Short Technical Reports, Biotechniques, vol. With a cell-free gap prepared, optical microscopy can be used to observe cells migrating into the wound area. [15] K. A. Discover a faster, simpler path to publishing in a high-quality journal. Inhibitors of cell invasion and metastasis can be effectively screened using the wound healing assay. 13 0 obj
Finally, the algorithm also considers cases in which the wound is not vertical but made at a specific angle. Competing interests: The authors have declared that no competing interests exist. Figure 3 | Methods to quantify cell migration in the wound healing assay. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. It allows the analysis of bright field, phase contrast, and fluorescence images. <>/F 4/Dest[ 17 0 R/XYZ 68 316 0] /StructParent 8>>
An increase in the threshold value increases the determined area (S3B Fig). Time lapse (0, 12, 18 and 24 hours. #C? In this regard, in S5 Fig we performed wound area measurements of images taken under different conditions (i.e., light, contrast, type of microscope), to find the range in which each of the parameters should be varied to identify the proper scratch area. The small difference found between the area estimated with the MiToBo plugin and our plugin validates the robustness of our approach. The most remarkable advantages of this assay are the low requirements of specialized equipment or expensive reagents, which makes it adaptable to research groups with limited budgets. List of parameters for the collected images. In addition to measuring changes in the wound area, the wound healing assay can also be applied to tracking the movement of individual cells at the leading edge of the wound. 59, pp. This is possible due to the reduced movement of the pipette tip enabled by the mold, which prevented inclination and helped to control the velocity and the pressure applied to make the scratch. endobj
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Citation: Suarez-Arnedo A, Torres Figueroa F, Clavijo C, Arbelez P, Cruz JC, Muoz-Camargo C (2020) An image J plugin for the high throughput image analysis of in vitro scratch wound healing assays. The image analysis allowed us to validate that the plugin can be useful in images collected in a wide variety of instruments in contrast to some macros that only work to identify the scratch from images collected with a limited number of instruments [25]. xn8xi
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,OnqTvuLKI+r/r&Q$4ed3? To do this, we initially enhanced the contrast of the image to increase the variance within the cell monolayer and facilitate its posterior binarization. Migr., vol. Examples of some of these applications are presented below. Accordingly, it is advisable to test two or three images with different combinations of the parameters before starting the analysis in order to localize an appropriate area of analysis such that the scratch can be fitted appropriately. The migration images can then be used to collect measurements or be visually scored [4]. <>/ExtGState<>/XObject<>/ProcSet[/PDF/Text/ImageB/ImageC/ImageI] >>/MediaBox[ 0 0 595.32 841.92] /Contents 64 0 R/Group<>/Tabs/S/StructParents 20>>
These time points should be collected within 24 hours of the experiment being started to minimize the confounding effects of cell replication on gap closure. (2020) used the assay to investigate novel hydrogel blends as a potential wound dressing. This plugin is useful for analyzing wound images since it relies on user-defined input values for the neighborhood radius of the variance filter, the threshold value for binarization, and the saturation percentage in the contrast enhancement, which can vary depending on the analyzed image. 5 0 obj
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Accurate measurements can be manually acquired using a digital camera fitted to the microscope; however, this process is time-consuming, and maintaining the same field-of-view along each gap can be difficult [4]. Therefore, the analysis of large datasets remains a bottleneck with many assays. endobj
This has been attributed to the components of the medium, which include multiple growth factors such as Transforming growth factor-beta one (TGF-1), Epidermal growth factor (EGF), Fibroblast growth factor (FGF), Hepatocyte growth factor (HGF), Interleukin 6 and 8 (IL-6 and IL8), as well as components of the extracellular matrix such as type I collagen and fibronectin [10]. Learn about all CytoSMART imaging solutions here. Some of these molecules include growth factors, proteins, natural compounds, and small pharmacological principles, among many others [2, 8, 9]. We determined the scratch area, wound coverage of total area, and average and standard deviation of the scratch width with the aid of our plugin. 9 0 obj
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According to Fig 6, there is no significant difference between the average width calculated with our plugin or calculated manually. It can be used with different operating systems and it is easy to install. As collective cell migration is linked to many physiological and pathological processes related to embryogenesis, wound repair, and cancer metastasis, the wound healing assay is broadly applicable [4], [8]. 3, pp. This monolayer represents the in vivo Yes 8 0 obj
Studies have also examined the migration of vascular smooth muscle cells [5], [6]. It is easy to use and can be adapted to analyze both individual images and stacks. Alves et al. [19] X. Wang, C. C. Decker, L. Zechner, S. Krstin, and M. Wink, In vitro wound healing of tumor cells: inhibition of cell migration by selected cytotoxic alkaloids, BMC Pharmacol. Moreover, these figures show that the exposure to hAdMSCs conditioned medium significantly reduced the scratch area of the wound (Fig 4B) after 4 hours compared with control medium. (Eq 3). %
These two methods can be time-consuming when performed manually. endobj
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Additionally, we convert the image resulting from the variance filter to a mask by applying the given threshold. Some phases that occur during wound healing have also been observed during cancer invasion [2, 3]. <>/F 4/Dest[ 29 0 R/XYZ 68 748 0] /StructParent 14>>
Additionally, our plugin allowed us to compare easily the two methods evaluated to make the wounds. B. endobj
Click through the PLOS taxonomy to find articles in your field. 15 0 obj
conditions of the tissue before wounding such as, an intact epithelium. Growth factors and matrix signals that determine the function of cells in regeneration processes orchestrate this behavior [1]. The wound healing assay can be used in the study of wound dressings. stream
Three-dimensional collective migration takes place on a tissue scaffold, and it is the movement of cells organized in a multicellular network of strands. Red lines of 0.1 mm represent the edges cut by the laser cutter, while Blacklines of 0.1 mm delineate regions for engraving. 2016 [2]. endobj
Cell proliferation can compete with cell migration to fill the gap made during the assay. Copyright: 2020 Suarez-Arnedo et al. In this context, the in vitro scratch wound healing assay is useful to evaluate the proliferation and migration process when exposing cells to metabolites present in the conditioned media. Autom., vol. Effect of changing the variance window radius while maintaining constant the threshold value = 100 and the percentage of saturated pixels = 0.01. Moreover, we also consider the standard deviation of the calculated widths to assess the homogeneity of the scratch and subsequently its closure. All the measurements and parameters were taken for 28 hours [n = 4 replicas per time for HaCaT exposed to hAdMSCs conditioned medium and n = 2 replicas per time for HaCaT exposed to control medium]. In this regard, the scratch or migration assay is a widely used tool for in vitro studies of the rates of migration [5], angiogenesis [6], movement [7], proliferation [2], and healing in response to different novel drug candidates. endobj
The radius must be big enough, so that the noise variance has no impact on tissue variance. Migration is considered the rate-limiting process during healing, and therefore migration assays are a key part of investigating wound healing [2]. Scale bar = 27550 m) images of wound healing closure in HaCaT exposed to hAdMSCs conditioned medium. With respect to Fig 5C, after enhancing image contrast, our plugin (variance-based method), estimated an area similar to that obtained with the ScratchAssayAnalyzer in MiToBo (only -0.51%2.8 of difference). <>
The HaCaT [26] cell line was provided by the Basic Medical Sciences Laboratory of the Faculty of Medicine of the Universidad de los Andes. W*];2Whh mxWKY>LZ^2%Qs|~0w'fB\(k+vg`v!=^.+uHEqiW_#
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The manual extraction of data is very time-consuming, subjective, and prone to error. Proliferation inhibitors, such as mitomycin C, can also be added to the medium. over time expressed as a percentage and is most often applied in live-cell imaging software [1], [12]. Yes This mold is reusable and can be sterilized using different strategies such as supercritical carbon dioxide, hydrogen peroxide gas plasma, ethylene oxide, and even -irradiation [31, 32]. endobj
We calculate the Euclidean distance between paired pixels, and we determine their average to describe wound width.