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In cattle, the endometrium during diestrus and early pregnancy displays cellular responses that are consequences of prior, transient stimuli. Goal was to establish a model to study cellular memory in the endometrium. The hypothesis is that stimuli given to endometrium in vivo are retained as a cellular memory that remains after bovine uterine epithelial cells (BUECs) are isolated, cultured, and further stimulated in vitro. Objectives were to measure BUEC proliferation/migration and responsiveness to recombinant bovine Interferon-tau (rbIFNT) in vitro: among cows that showed estrus (experiment 1 [Exp1]), cows that became or not pregnant to artificial insemination (Exp2), cows that received or not supplemental progesterone (P4; Exp3) and cows that received or not a COX-1/2 inhibitor (Exp4). Only cows that displayed estrus were included in studies. For all experiments endometrial cytology was collected 4 days after estrus, BUECs were cultured, propagated, and submitted to rbIFNT treatment and an in vitro scratch assay. In Exp1, different cows spontaneously grouped according to proliferative/migratory capacity and responsiveness to rbIFNT of their respective BUECs. In Exp2, BUECs from pregnant cows showed greater rbIFNT responsiveness and cellular proliferation. In Exp3, BUECs from cows supplemented with P4 presented inhibited proliferation and increased expression of RSAD2. In Exp4, Flunixin Meglumine modified rbIFNT responsiveness of BUECs in an IFN-signaling pathway-specific manner. In conclusion, physiological and pharmacological stimuli received by the endometrium in vivo were retained as cellular memory in BUECs, persisted in culture, and changed BUEC proliferation/migration and responsiveness to rbIFNT, which are characteristics associated with fertility in cattle. Summary Sentence Endometrial cell functions associated with the pregnancy outcome are regulated by past events whose effects are retained as cellular memories. Graphical Abstract

The wound‐scratch assay is a widely used in vitro model for studying collective cell migration, a fundamental process contributing to wound closure and re‐epithelialisation. Owing to its simplicity, low cost, and adaptability, it has become a foundational tool for early‐stage wound‐healing research and therapeutic screening. The assay involves generating a defined gap in a confluent cell monolayer and monitoring gap closure over time as a surrogate readout of repair. This narrative review examined 199 published studies, identifying 73 relevant to wound healing. A technical hierarchy of wound creation methods was identified across three main categories: mechanical approaches (e.g., pipette tips and cell scrapers), accessible but prone to operator‐dependent variability; semi‐automated systems (e.g., inserts and wound maker devices), which improve reproducibility; and fully‐automated robotic platforms offering high precision and high‐throughput capability. While these advances enhance technical consistency, they do not overcome the assay's fundamental biological constraints. Importantly, gap closure in the wound‐scratch assay primarily reflects planar collective cell migration and does not recapitulate the integrated inflammatory, vascular, metabolic, and extracellular matrix‐dependent processes that govern wound repair in vivo. Consequently, bioactive compounds acting through antioxidant, anti‐inflammatory, angiogenic, or matrix‐modulating pathways may have their therapeutic potential underestimated or misclassified when assessed using migration‐only readouts. Preliminary in‐house (unpublished) data are presented to illustrate this limitation, demonstrating modest migration effects for compounds with established wound‐healing activity in vivo. Despite these limitations, the wound‐scratch assay remains a valuable first‐line, hypothesis‐generating tool when interpreted appropriately, with future utility dependent on integration with adapted models and complementary assays for translation. Key Messages The wound‐scratch assay is a simple, cost‐effective, and widely used in vitro model for quantifying collective cell migration, a fundamental component of wound closure. Wound creation methodologies can be broadly classified as mechanical, semi‐automated, or fully automated, each offering distinct trade‐offs in accessibility, reproducibility, and biological fidelity. While valuable for early‐stage mechanistic insight and preliminary therapeutic screening, the assay does not recapitulate the cellular, inflammatory, vascular, and matrix complexity of in vivo wound healing. Strategic technical refinement and integration with complementary in vitro and ex vivo models are essential to enhance reproducibility, interpretability, and translational relevance in wound‐healing research.

Cell migration is a key procedure involved in many biological processes including embryological development, tissue formation, immune defense or inflammation, and cancer progression. How physical, chemical, and molecular aspects can affect cell motility is a challenge to understand migratory cells behavior. assays are excellent approaches to extrapolate to situations and study live cells behavior. Here we present four protocols that describe step-by-step cell migration, invasion and adhesion strategies and their corresponding image data quantification. These current protocols are based on wound healing assays (comparing traditional pipette tip-scratch assay vs. culture insert assay), 2D individual cell-tracking experiments by live cell imaging and spreading and transwell assays. All together, they cover different phenotypes and hallmarks of cell motility and adhesion, providing orthogonal information that can be used either individually or collectively in many different experimental setups. These optimized protocols will facilitate physiological and cellular characterization of these processes, which may be used for fast screening of specific therapeutic cancer drugs for migratory function, novel strategies in cancer diagnosis, and for assaying new molecules involved in adhesion and invasion metastatic properties of cancer cells.

Proliferation and migration of keratinocytes and fibroblasts play an important role in cutaneous wound healing, while oral mucosal squamous cell proliferation and migration are crucial for oral wound healing. In this study, the phytochemical profile of Pluchea indica branch ethanolic extract was characterized. The bioactive compound of Pluchea indica branch ethanolic extract was identified and analyzed by the validated HPLC method. The nanoparticles of P. indica branch extract were formulated by solvent displacement method to increase the solubility and the colloidal stability of the extract. The stability of the nanoparticles was investigated by using the dynamic light scattering technique. Effects of P. indica crude extract and nanoparticles on cell viability, proliferation and migration of primary epidermal keratinocytes, human dermal fibroblasts, and oral mucosal keratinocyte cells were investigated by MTT assay and scratch assay, respectively. The results showed that P. indica branch extract contained a high content of total phenolic and total flavonoids. The HPLC analysis revealed that the main compound in the extract was 4,5-O-dicaffeoylquinic acid. The cell viability of the extract and nanoparticles decreased when cells were exposed to a high concentration of extract and nanoparticles. These results demonstrate that P. indica branch extract and extract nanoparticles at specific concentrations possess in vitro wound healing activity and they may be possibly used to treat different types of wounds including dermal and oral mucosal wounds.

Cell migration plays a central role in tumor progression and metastasis, making it a critical parameter in both cancer biology and therapeutic evaluation. A range of in vitro migration assays are commonly used to assess treatment-induced effects on motility, each with distinct advantages and limitations that must be considered when interpreting results. In this study, we systematically compared four migration assay formats—scratch, zone-exclusion (Z-E), transwell, and single-cell tracking—across five melanoma cell lines treated with the multi-kinase inhibitor nintedanib. Migration inhibition was quantified for each assay, alongside machine learning-based classification to evaluate the discriminative power of assay-derived features in detecting treatment effects. All assays detected inhibitory effects of nintedanib, although response magnitude varied across formats. The transwell assay demonstrated clear inhibition in most lines but was sensitive to morphological changes. Gap closure assay-based migration rankings were influenced by cell size, underscoring the importance of size normalization. Scratch assays exhibited greater variability and potential confounding due to cell damage, while Z-E assays generated more uniform gaps without mechanical disruption. Single-cell tracking enabled high-resolution phenotypic profiling, including cell-specific features such as size and shape, resulting in consistently high classification accuracy. These findings highlight the importance of selecting appropriate assay formats to accurately evaluate migration behavior and the therapeutic efficacy of anti-migratory compounds.

Wound healing is essential for the restoration of the barrier function of the skin. During this process, cells at the wound edges proliferate and migrate, leading to re-epithelialization of the wound surface. Wound healing assays are used to study the molecular mechanisms of wound repair, as well as in the investigation of potential therapeutics and treatments for improved healing. Numerous models of wound healing have been developed in recent years. In this review, we focus on in vitro assays, as they allow a fast, cost-efficient and ethical alternative to animal models. This paper gives a general overview of 2-dimensional (2D) cell monolayer assays by providing a description of injury methods, as well as an evaluation of each assay’s strengths and limitations. We include a section reviewing assays performed in 3-dimensional (3D) culture, which employ bioengineered skin models to capture complex wound healing mechanics like cell-matrix interactions and the interplay of different cell types in the healing process. Finally, we discuss in detail available software tools and algorithms for data analysis.

Wound healing is a complex process that can be improved through advanced biomedical approaches. Incorporating nanopolymers and plant extracts into wound dressings offers a favorable strategy for promoting tissue repair. Nanopolymers provide a controlled environment for sustained drug release while also protecting the wound from external contaminants. When combined with bioactive compounds from plant extracts, which possess antioxidant, anti-inflammatory, and antimicrobial properties, this hybrid approach can accelerate healing, reduce infection, and improve tissue regeneration. Hence, in this study, we have synthesized alginate/gelatin hydrogel blended with only nanosilver (Alg/gel-Ag) and with nanosilver and plant extracts like aloe vera, curcumin, plantain peel extract, and Calendula flower petal extract (Alg/gel-AgP). The synthesized hydrogels were characterized using different photophysical tools, and the cytotoxicity effect was studied using a fibroblast cell line (V79). The antibacterial effect of the hydrogels was also observed against E. coli and S. aureus, determining the MIC and MBC. The wound healing in vitro was also assessed using scratch assay which depicted a rapid wound closure for Alg/gel-AgP compared to the untreated control and Alg/gel-Ag. The combined effect between nanotechnology and natural extracts represents a novel and effective approach for enhancing the wound healing process.

Human glioblastoma multiforme (GBM) is one of the most malignant brain tumors, with a high mortality rate worldwide. Conventional GBM treatment is now challenged by the presence of the blood–brain barrier (BBB), drug resistance, and post-treatment adverse effects. Hence, developing bioactive compounds isolated from plant species and identifying molecular pathways in facilitating effective treatment has become crucial in GBM. Based on pharmacodynamic studies, andrographolide has sparked the interest of cancer researchers, who believe it may alleviate difficulties in GBM therapy; however, it still requires further study. Andrographolide is a bicyclic diterpene lactone derived from Andrographis paniculata (Burm.f.) Wallich ex Nees that has anticancer properties in various cancer cell lines. The present study aimed to evaluate andrographolide’s anticancer effectiveness and potential molecular pathways using a DBTRG-05MG cell line. The antiproliferative activity of andrographolide was determined using the WST-1 assay, while scratch assay and clonogenic assay were used to evaluate andrographolide’s effectiveness against the cancer cell line by examining cell migration and colony formation. Flowcytometry was also used to examine the apoptosis and cell cycle arrest induced by andrographolide. The mRNA and protein expression level involved in the ERK1/2/c-Myc/p53 signaling pathway was then assessed using qRT-PCR and Western blot. The protein–protein interaction between c-Myc and p53 was determined by a reciprocal experiment of the co-immunoprecipitation (co-IP) using DBTRG-05MG total cell lysate. Andrographolide significantly reduced the viability of DBTRG-05MG cell lines in a concentration- and time-dependent manner. In addition, scratch and clonogenic assays confirmed the effectiveness of andrographolide in reducing cell migration and colony formation of DBTRG-05MG, respectively. Andrographolide also promoted cell cycle arrest in the G2/M phase, followed by apoptosis in the DBTRG-05MG cell line, by inducing ERK1/2, c-Myc, and p53 expression at the mRNA level. Western blot results demonstrated that c-Myc overexpression also increased the production of the anti-apoptotic protein p53. Our findings revealed that c-Myc and p53 positively interact in triggering the apoptotic signaling pathway. This study successfully discovered the involvement of ERK1/2/c-Myc/p53 in the suppression of the DBTRG-05MG cell line via cell cycle arrest followed by the apoptosis signaling pathway following andrographolide treatment.

Iron oxide nanoparticles (IONPs) were synthesized from the Proteus vulgaris ATCC-29905 using the extracellular methodology. The UV–Vis spectrophotometry showed the λ-max (maximum absorbance) absorption peak at 310 nm. The FTIR analysis showed amides and other functional groups are associated with IONPs. The TGA results showed less protein loss (ΔY = 9.045%) at a protein degradation temperature of 71.73 °C. The FESEM images showed particles are spherical in shape. The EDX confirms the presence of iron. Zeta potential interface was found to be 79.5 mV that confirms its stability. The TEM results showed particles are spherical with a diameter between 19.23 nm ad 30.51 nm. These IONPs showed good antibacterial activity and also showed good activity against methicillin resistant staphylococcus aureus (MRSA). It showed good antioxidant activity. These IONPs exhibits good cytotoxic effect against U87 MG—glioblastoma cancer cells, showed IC50 value at 250 µg/ml compared with healthy L-132 cells. Scratch assay showed IONPs inhibit the cell migration of the HT-29 cancer cells. The nanoparticles synthesized from bacteria are safe and non-hazardous. It was expected that these IONPs could become a potential anticancer and antibacterial agent, and in the future it opens a new path for treating the cancer patients.

This study aims to investigate the effectiveness of low power red light (661 nm) in accelerating the wound healing process of an in vitro scratch assay model of keratinocytes. Furthermore, the study aims to clarify the role of light irradiation parameters, optimize them and gain additional insight into the mechanisms of wound closure as a result of photobiomodulation. Wound healing was studied using scratch assay model of NCTC 2544 keratinocytes. Cells were irradiated with a laser at various power densities and times. Images were acquired at 0, 24, 48 and 72 h following the laser treatment. Cellular proliferation was studied by MTT. ROS were studied at 0 and 24 h by fluorescence microscopy. Image analysis was used to determine the wound closure rates and quantify ROS. The energy range of 0.18–7.2 J/cm 2 was not phototoxic, increased cell viability and promoted wound healing. Power and irradiation time proved to be more important than energy. The results indicated the existence of two thresholds in both power and irradiation time that need to be overcome to improve wound healing. An increase in ROS production was observed at 0 h only in the group with the lowest healing rate. This early response seemed to block proliferation and finally wound healing. Low level laser light at 661 nm enhanced both proliferation and migration in keratinocytes, providing evidence that it could possibly stimulate wound healing in vivo. The observed results are dependent on irradiance and irradiation time rather than energy dose in total.