Cell Biology

Osteoclasts are terminally differentiated multinucleated giant cells that mediate bone resorption and regulate skeletal homeostasis under physiological and pathological states. Excessive osteoclast activity will give rise to enhanced bone resorption, being responsible for a wide range of metabolic skeletal diseases, ranging from osteoporosis and rheumatoid arthritis to tumor-induced osteolysis. Therefore, the construction of in vitro models of osteoclast-mediated bone resorption is helpful to better understand the functional status of osteoclasts under (patho)physiological conditions. Notably, it is essential to provide an in vivo–relevant bone substrate that induces osteoclasts to generate authentic resorption lacunae and excavate bone. Here, we summarize the experimental design of a reproducible and cost-effective method, which is suitable for evaluating the regulatory mechanisms and influence of molecular agonists and antagonists as well as therapeutics on osteoclast-mediated bone-resorbing activity.

The parasympathetic nervous system is essential for salivary gland development and functionality. Parasympathetic neuron (parasymN) innervation is the main neural network that controls salivary secretion. Therefore, an exclusive model to study parasympathetic neurons and salivary gland tissue circuitry will significantly improve the understanding of the role of parasymN activation on salivary regulation. Harvesting primary rodent parasymNs is challenging due to their body-wide disbursed location. Similarly, the salivary glands are distributed in various locations around and within the oral cavity. Here, we present a coculture model system using human pluripotent stem cell (hPSC)-derived parasymNs and primary mouse von Ebner’s gland cells. We previously reported the first protocol to robustly generate human parasymNs from hPSCs through the Schwann cell precursor (SCP) lineage. The hPSC-parasymNs are functional and have been applied to model several autonomic disorders. We also used a Sox10-Cre::tdTomato (hereafter referred to as RFP) reporter mouse line, which labeled von Ebner’s glands, a type of minor salivary gland connected to the trough of circumvallate and foliate taste papillae. This labeling allowed for visualization and efficient isolation of primary tissues in young adult mice (8–10 weeks). By coculturing the two tissues, human parasymNs control mouse salivary gland cell growth and activation. Both parasymNs and primary salivary gland cells can be frozen and stocked at early stages of differentiation and isolation, making applications easier. This novel coculture model system could also be used to model and study related human diseases in the future, such as dry mouth syndrome.

Plants use CO₂, water, and light energy to generate carbohydrates through photosynthesis. During daytime, these carbohydrates are polymerized, leading to the accumulation of starch granules in chloroplasts. The catabolites produced by the degradation of these chloroplast starch granules are used for physiological responses and plant growth. Various staining methods, such as iodine staining, have previously been used to visualize the accumulation of chloroplast starch granules; however, these staining methods cannot be used to image live cells and/or provide confocal images with non-specific signals. In this study, we developed a new imaging method for the fluorescent observation of chloroplast starch granules in living plant cells by staining with fluorescein, a widely available fluorescent dye. This simple staining method, which involves soaking a leaf disk in staining solution, shows high specificity in confocal images. Fluorescent images of the stained tissue allow the cellular starch content of living cells to be quantified with the same level of accuracy as a conventional biochemical method (amyloglucosidase/α-amylase method). Fluorescein staining thus not only enables the easy and clear observation of chloroplast starch granules but also allows for precise quantification in living cells.

Drug-induced hearing injury (ototoxicity) is a common, debilitating side effect of many antibiotic regimens that can be worsened by adverse drug interactions. Such adverse drug interactions are often not detected until after drugs are already on the market because of the difficulty of measuring all possible drug combinations. While in vivo mammalian assays to screen for ototoxic damage exist, they are currently time-consuming, costly, and limited in throughput, which limits their utility in assessing drug interaction outcomes. To facilitate more rapid quantification of ototoxicity and assessment of adverse drug interactions that impact ototoxicity, we have developed a high-throughput workflow we call parallelized evaluation of protection and injury for toxicity assessment (PEPITA). PEPITA uses zebrafish larvae to quantify ototoxic damage and protection. Previous work has shown that hair cells (HCs) in the zebrafish lateral line are very similar to human inner ear HCs, meaning zebrafish are a viable model to test drug-induced ototoxicity. In PEPITA, we expose zebrafish larvae to different combinations of drugs, fluorescently label the HCs, and subsequently use microscopy to quantify the brightness of the fluorescently labeled HCs as an assay for ototoxic damage and hair-cell viability. PEPITA is a reproducible, low-cost, technically accessible, and high-throughput assay. These advantages allow many experiments to be conducted in parallel, paving the way for systematic evaluation of drug-induced hearing injury and other multidrug interactions.

This paper presents a refined, user-friendly protocol for using boron-dipyrromethene (BODIPY) to assess and quantify foam cells and lipid droplet–accumulating microglia (LDAM) in mouse brain tissue. The protocol aims to enhance existing methodologies by offering precise and efficient evaluation of foam cells and LDAM burden in various neuropathological conditions linked to lipid metabolism and neuroinflammation. A notable challenge in analyzing tissue from mouse models of these neurodegenerative disorders is the interference caused by the autofluorescent molecule lipofuscin. Our protocol addresses this issue with specific steps that effectively distinguish BODIPY fluorescence from lipofuscin autofluorescence, using advanced imaging techniques and filter settings to ensure accurate and reliable analysis. By providing a straightforward and accessible method, this research aims to facilitate the broader adoption of BODIPY-based techniques for detailed foam cell and LDAM analysis in mouse brain tissue, potentially enhancing diagnostic capabilities and deepening our understanding of how these cells contribute to neurodegenerative disease mechanisms.

Dengue virus (DENV), a common and prevalent mosquito-borne endemic disease, is caused by four serotypes (DENV-1–4) and has spread rapidly on a global scale over the past decade. A crucial step in the development of antiviral therapeutics requires the utilization of in vitro cell-based techniques, such as plaque assays and focus-forming assays (FFA) for virus quantification. Vero cells have been widely used for FFA and plaque assay; however, there are instances when their efficacy and efficiency in the detection of certain clinical DENV isolates are low. Here, we showed that BHK-21 cells are more sensitive than Vero cells in the detection of all DENV-1–4 plaques and foci. In addition, we developed an improved FFA protocol for the quantification of all four DENV serotypes. Using a pan-flavivirus envelope (E) antibody, we reduce the possibility of false positives by defining a focus to consist of a minimum of eight infected cells. We outlined a protocol using the Operetta® high-content imaging system to automate the digital capture of these infected cells. A pipeline was also designed using the CellProfilerTM automated image analysis software to detect these foci. We then compare the results of the improved FFA with plaque assay. Notably, the improved FFA detected clear foci of the DENV-4 strain that does not form distinct plaques. We subsequently demonstrated the potential application of the improved FFA protocol in antiviral testing, utilizing a nucleoside inhibitor of DENV, NITD008 as a control. The protocol is amenable to a diverse array of applications, including high-throughput compound screening (HTS).

The adoptive transfer of autologous, long-lived, gene-repaired T cells is a promising way to treat inherited T-cell immunodeficiencies. However, adoptive T-cell therapies require a large number of T cells to be manipulated and infused back into the patient. This poses a challenge in primary immunodeficiencies that manifest early in childhood and where only small volumes of blood samples may be available. Our protocol describes the ex vivo expansion of potentially long-lived human T memory stem cells (T_SCM), starting from a limited number of peripheral blood mononuclear cells (PBMCs). Using the perforin gene as an example, we provide detailed instructions for precise gene repair of human T cells and the expansion of T_SCM. The efficiency of precise gene repair can be increased by suppressing unintended non-homologous end-joining (NHEJ) events. Our protocol yields edited T-cell populations that are ready for phenotyping, genome-wide off-target analysis, and functional characterization.

MreB is a prokaryotic actin homolog. It is essential for cell shape in the majority of rod-shaped cell-walled bacteria. Structural and functional characterization of MreB protein is important to understand the mechanism of ATP-dependent filament dynamics and membrane interaction. In vitro studies on MreBs have been limited due to the difficulty in purifying the homogenous monomeric protein. We have purified MreB from the cell-wall-less bacteria Spiroplasma citri, ScMreB5, using heterologous expression in Escherichia coli. This protocol provides a detailed description of purification condition optimization that led us to obtain high concentrations of stable ScMreB5. Additionally, we have provided a protocol for detecting the presence of monovalent ions in the ScMreB5 AMP-PNP-bound crystal structure. This protocol can be used to obtain a high yield of ScMreB5 for carrying out biochemical and reconstitution studies. The strategies used for ScMreB5 show how optimizing buffer components can enhance the yield and stability of purified protein.

The mammary gland undergoes functional, developmental, and structural changes that are essential for lactation and reproductive processes. An overview of such unique tissue can offer clearer insights into mammary development and tumorigenesis. Compared to traditional methods, mouse mammary gland whole mount is a pivotal technique that provides three-dimensional structural perspectives on gland morphology and developmental stages, offering an inexpensive and accessible approach. This protocol outlines the tissue isolation of the mouse mammary gland and provides detailed instructions for whole-mount staining and analysis. Mammary gland tissues are carefully dissected from euthanized mice and stained with Carmine Alum to highlight the ductal structures, enabling detailed visualization of the branching patterns and morphological changes. Light microscopy is used to capture a panoramic image of the stained mammary gland, enabling the quantitative analysis of terminal end buds (TEBs) and bifurcated TEBs to further investigate mammary gland remodeling. This method can provide invaluable insights, particularly in the study of mammary gland morphogenesis and tumorigenesis, underscoring its significance in both basic research and clinical applications.

Endometrial cancer (EC) is the leading cause of gynecologic cancer morbidity and mortality in the U.S. Despite advancements in cancer research, EC death rates are increasing, particularly high-grade endometrial cancers. The development of three-dimensional (3D) patient-derived organoid (PDO) models for EC is crucial, as they provide a more accurate representation of the biological and genetic complexity of a patient’s tumor compared to traditional 2D cell lines. Here, we describe a protocol for cultivating PDO models from normal endometrium and EC across different EC subtypes. These EC PDO models can be expanded across multiple passages and facilitate the exploration of tumor behavior and drug responses, thereby advancing our understanding of the disease and potentially leading to more effective and individualized novel therapeutic strategies.