细胞生物学

The integrity of epithelial barriers is essential for maintaining tissue homeostasis, particularly in the intestinal tract, where it separates the host from the complex luminal environment. Two complementary, standard methods for assessing this barrier are transepithelial electrical resistance (TEER), which provides a rapid, non-destructive measure of ionic conductance across tight junctions, and the fluorescein isothiocyanate (FITC)-dextran assay, which directly quantifies paracellular macromolecule flux. This protocol details a robust and reproducible method for performing both assays using intestinal epithelial cell monolayers (e.g., Caco-2, T84) cultured on permeable Transwell supports. We outline the procedure from cell culture and monolayer differentiation to TEER measurement with an Epithelial Volt/Ohm Meter 3 (EVOM3) and the subsequent FITC-dextran permeability assay. By combining these techniques, this protocol provides a comprehensive assessment of barrier function, making it ideal for studying tight junction dynamics and regulation under various experimental conditions, such as cytokine stimulation, drug screening, or microbial challenges.

Stochastic optical reconstruction microscopy (STORM) is a single-molecule localization microscopy technique that enables visualization of cellular structures beyond the diffraction limit. This approach has revealed previously inaccessible ultrastructural details in a wide range of cellular components, including the actin cytoskeleton, clathrin-coated pits, mitochondria, and bacterial nucleoid-associated proteins. STORM relies on the sequential emission of single photons from photosensitive fluorophores, which are precisely localized before entering a dark state or undergoing photobleaching. By activating fluorophores individually and fitting their point spread functions (PSFs), the center of mass can be calculated with a localization precision of up to ~20 nm. The parallel detection of thousands of single-molecule events, each assigned to distinct spatial coordinates, enables the reconstruction of a high-resolution image. Here, we describe a simple and efficient STORM workflow—including sample preparation, image acquisition, and quality control measurements—that we used to visualize various subcellular structures, such as mitochondria, microtubules, and lysosomes labeled with the commonly employed cyanine dye Alexa Fluor 647, as well as the actin cytoskeleton stained with Alexa Fluor 488–conjugated phalloidin. Image acquisition was performed using a conventional epifluorescence/total internal reflection (TIRF) microscope adapted for STORM imaging. Key adaptations included the use of a 160×/1.43 NA oil-immersion objective and a high-power mode, which concentrates the laser beam onto a small region of the sample, ensuring sufficient light intensity to drive fluorophores into the dark state. In addition, implementing a 1.6× magnification lens and a 4×4 binning camera mode allowed us to achieve a 100-nm pixel size optimal for reliable molecule detection. We believe that this protocol will be highly valuable to the microscopy community, as it lowers technical barriers to performing STORM on widely available microscopy platforms, thereby facilitating broader implementation of this powerful super-resolution technique.

Epigenetic modifications play essential roles in regulating gene expression and maintaining cellular identity. Accumulating evidence suggests that chemical agents can contribute to carcinogenesis through epigenetic alterations, such as changes in DNA methylation and histone modifications, even in the absence of direct DNA damage. Here, we have developed a simple, cost-effective, and quantitative reporter assay, termed the epi-TK assay, to evaluate chemically induced epigenetic alterations. The assay is built upon the thymidine kinase (TK) gene mutation assay, a standardized and widely used in vitro genotoxicity assay for chemical safety evaluation. This system is based on an engineered human lymphoblastoid cell line (mTK6), in which the promoter region of the endogenous housekeeping TK gene is site-specifically methylated using epigenome-editing technology, resulting in stable transcriptional repression. Following chemical exposure, epigenetic perturbations at the TK locus are detected by culturing cells under hypoxanthine–aminopterin–thymidine selection and quantifying the frequency of TK revertant colonies, which reflects restoration of TK gene expression. Using the DNA methyltransferase 1 inhibitor GSK3484862 as a model compound, this protocol demonstrates that the epi-TK assay enables sensitive and quantitative detection of epigenetic state transitions. Importantly, this assay allows bi-directional detection of epigenetic changes, including DNA demethylation events and broader alterations in histone modification landscapes. Together, the epi-TK assay provides a practical and quantitative platform for evaluating epigenetic toxicity, with potential applications in chemical safety assessment frameworks.

Natural killer (NK) cells are crucial innate immune effectors, mediating cytotoxicity against cancer and infected cells through receptors such as NKG2D. Reliable quantification of NK cell subsets is essential for evaluating NK cell-based immune responses in cancer research. Unlike other assays, including traditional flow cytometry used in assessing NK cells, imaging flow cytometry (IFC) is a simple and direct method for quantitative analysis of NK cells. This protocol describes the necessary procedures, including harvesting splenocytes, acquiring these cells labeled with NKG2D antibodies, and analyzing IFC data with IDEAS^® software. We applied this protocol to quantitatively assess the number of splenic NKG2D⁺ NK cells in mice injected with SVTneg2 cancer cells (which carry the p53 G242A missense mutation) and compared them to mice injected with EMT6 cancer cells (which have wild-type p53) or normal fibroblasts. We found that the SVTneg2 cancer cells significantly decreased the number of NKG2D⁺ NK cells in mice by approximately 2-fold (933 cells vs. 2360 cells, p < 0.001) compared with mice injected with EMT6 cancer cells. This IFC protocol can be applied to directly quantify NK cells in vivo. This quantitative protocol allows novices to quickly handle the analysis of cytotoxic NK cells with a single NKG2D marker. Further multicolor flow cytometry and cytokine assay may be required to precisely define the subtypes and effects of NK cells in anticancer immunity.

Aloe vera has long been used for its diverse pharmacological properties, motivating continued interest in isolating and preserving the bioactive molecules responsible for its therapeutic potential. More recently, Aloe vera–derived extracellular vesicles (Av-EVs) have emerged as nanoscale, cell-free carriers capable of retaining and delivering these properties, making them attractive for various biomaterials, nanomedicine, and regenerative medicine applications. Multiple techniques are available for extracellular vesicle isolation. These include ultracentrifugation, polymer-based precipitation, size-exclusion chromatography, immunoaffinity capture, ultrafiltration, density gradient separation, and emerging microfluidic platforms. Each method presents distinct trade-offs in purity, yield, scalability, and downstream compatibility. Despite this diversity, standardized workflows tailored to Av-EV isolation remain limited, and the influence of homogenization-induced shear forces and plant maturity on vesicle recovery and characterization has not been systematically addressed. Here, we present a reproducible protocol for isolating Av-EVs from Aloe vera gel employing two distinct homogenization strategies: manual, no-shear force (NB EVs), and blender-based shear-force homogenization (B EVs). The workflow covers gel preparation, serial centrifugation for debris removal, ultracentrifugation as the gold standard for vesicle enrichment, and final sterile filtration. This protocol enables consistent recovery of Av-EVs suitable for physicochemical characterization and functional analyses. It is simple and relies on commonly available laboratory equipment, facilitating broad adoption by ultracentrifugation users and offering adaptability to diverse research projects involving purified Aloe vera gel and Av-EVs, including studies focused on wound healing, fibrotic scarring, and regenerative processes, where coordinated antioxidant, anti-inflammatory, antimicrobial, immunomodulatory, and moisturizing responses are of interest.

We established a step-by-step approach for generating a single-nucleotide mutation in the promoter region of an immune regulatory gene in human monocyte THP-1 cells by employing a plasmid-based CRISPR-Cas9 system delivered via transfection with a homology-directed repair template DNA (HDR). Key steps include designing a single-guide RNA (sgRNA), cloning it into a CRISPR plasmid encoding the Cas9 protein, transfection of the plasmid constructs along with single-stranded oligonucleotide repair template (ssODNs) into THP-1 cells, followed by selection and validation. This approach provides a precise and relevant model to investigate the role of single polymorphisms in the regulation of inflammatory gene expression in human monocytes. In addition to the rs1024611 single-nucleotide polymorphism (SNP), this CRISPR/Cas9-based strategy is broadly applicable to functional studies of noncoding and coding variants in innate immune genes.

Reactive oxygen species (ROS) are central regulators of plant development and stress responses, with hydrogen peroxide (H₂O₂) acting as a key signaling molecule whose spatial distribution determines adaptive versus damaging outcomes. Accurate detection of H₂O₂ at tissue and cellular resolution is therefore essential for understanding redox-dependent regulation of plant growth. A variety of techniques have been used to monitor H₂O₂, including bulk spectrophotometric and fluorometric assays, genetically encoded sensors for real-time measurements, and chemical probes for in situ detection. While these approaches differ in sensitivity, specificity, and temporal resolution, many are limited by a lack of spatial information, technical complexity, or dependence on transgenic material. Here, we present a detailed protocol for 3,3′-diaminobenzidine (DAB)-based histochemical detection of H₂O₂ in seedling roots, covering staining, imaging, and semi-quantitative image analysis using open-source software (FIJI/ImageJ). The method relies on peroxidase-mediated oxidation of DAB, resulting in a stable, light-resistant, and insoluble precipitate that enables visualization of H₂O₂ accumulation with high spatial resolution. This protocol provides a robust, accessible, and genetically independent approach for spatial analysis of H₂O₂ in plant tissues. Its simplicity, compatibility with diverse genotypes and treatments, and suitability for semi-quantitative analysis make it a valuable tool for examining the spatial distribution of H₂O₂, thereby providing spatial insight into redox-related regulatory processes during plant development and stress responses.

Metastasis is initiated by cell invasion of the basement membrane, facilitating cell migration and colonization at a secondary tumor site. Cancer cells remodel the cytoskeleton to form ventral protrusions, termed invadopodia, that traffic and deliver matrix metalloproteases to degrade the extracellular matrix. Traditional efforts have utilized immunolabeling to measure protein localization within invadopodia, an approach limited by reduced temporal resolution, logistical challenges in orienting invadopodia within the focal plane of the objective lens, and impaired ability to reconstitute physiological conditions. Here, we describe a protocol for constructing and utilizing the axial invasion chamber (AIC) to perform live-cell 3D visualization of mature elongating invadopodia under physiological conditions. The AIC is simple to build, using standard 35 mm glass-bottom dishes that suit most microscope stage holders. A polyester membrane is used to uniformly orient and promote invadopodia formation and restrict cell migration. The AIC extracellular matrix is composed of readily available reagents that have been optimized to facilitate cell adhesion and invadopodia maturation. Critical advances of the AIC include imaging and measurements of protein localization without immunolabeling, imaging of live cell invadopodia using conventional inverted microscopes, and production of a fully operational apparatus within 28 h from initial assembly. While the protocol has been used for live-cell invadopodia protein localization and structure, it provides an opportunity to interchange components of the polyester membrane and/or the extracellular matrix to optimize the device for a variety of different cell types and cell invasion studies.

Patient-derived glioblastoma (GBM) cells are valuable models for GBM research due to their rarity and the highly lethal nature of this cancer. Preserving these cells through long-term cryopreservation is therefore essential for advancing future investigations. However, recent studies have reported that standard cell recovery protocols are inefficient, resulting in poor cell survival and limited regrowth. Here, we established an optimized culture protocol that enhances the recovery and expansion of patient-derived GBM cells by combining Matrigel with an increased concentration of fetal bovine serum (FBS). This approach significantly improves cell attachment and recovery after thawing cells that have been cryopreserved for more than a decade. Importantly, the recovered cells retain key phenotypic characteristics and remain suitable for downstream applications, including drug testing and spheroid formation. Together, this optimized protocol provides a novel strategy to increase the availability of patient-derived GBM cells by improving their efficient recovery from long-term cryopreservation, thereby maximizing their utility in GBM research.

Extracellular vesicles (EVs) are critical mediators of cell–cell communication and play a key role in male reproductive biology by modulating sperm function. This protocol describes a robust and reproducible workflow for isolating EVs from ram seminal plasma using size-exclusion chromatography (SEC) and assessing their uptake by ram spermatozoa. In contrast to ultracentrifugation-based methods, SEC provides a gentle and more efficient isolation approach that preserves EV integrity and functionality. A central innovation of this protocol is the use of carboxyfluorescein succinimidyl ester (CFSE)-labeled seminal plasma EVs (SP-EVs) to evaluate their incorporation into sperm cells through two complementary detection platforms: (i) flow cytometry with standard resolution and (ii) confocal microscopy, for spatial confirmation of EV–sperm interactions. By bridging the gap between EV isolation and functional analysis, this protocol provides a valuable tool for investigating the role of EV–cell interactions. Specifically, it offers potential applications in male fertility preservation, biomarker discovery, and the development of EV-based therapeutic strategies in reproductive medicine.