Cell Biology

Three-dimensional cell models, such as spheroids, represent a more physiological arrangement in which cells can grow, allowing them to develop cell–cell interactions in all dimensions. The most common methods for growing spheroids are scaffold-based, typically using either extracellular matrix or hydrogels as a physical support for the cellular assembly. One key problem with this approach is that the spheroids that are produced can be highly variable in size and shape. The protocol presented here allows for the systematic production of uniform spheroids in a short time frame by utilising a micropatterned plate. We show that spheroids can be used to investigate fundamental research questions, such as how the endomembrane system is organised in cells. Our protocol can be used in a manual or automated manner, potentially allowing scaling up for screening applications. Furthermore, without the complication of removing the spheroids from the extracellular matrix or hydrogel, as would be required in scaffold-based systems, spheroids can easily be used in other downstream applications.

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] is a phospholipid enriched on the cytoplasmic leaflet of the plasma membrane, where it plays important roles in membrane trafficking and cytoskeletal dynamics through proteins that directly bind to it. PI(4,5)P₂ can be metabolized to other phosphorylated forms of phosphatidylinositol to regulate numerous processes such as cell growth and development. PI(4,5)P₂ can also be hydrolyzed to generate the second messengers diacylglycerol (DAG) and inositol triphosphate (IP₃). Altered metabolism or mislocalization of PI(4,5)P₂ can perturb one or more of its functions and contribute to disease states. Here, we present a protocol to visualize and quantify the localization of PI(4,5)P₂ in live cells. The protocol uses a highly specific PI(4,5)P₂ protein binding domain coupled to enhanced green fluorescence protein (PH-PLCD1-GFP), enabling localization and quantification of cytosol-facing PI(4,5)P₂ to be determined. Localization and quantification of the PH-PLCD1-GFP, PI(4,5)P₂ specific probe, is enabled by fluorescence imaging and confocal microscopy. This approach can be used to study the dynamics of PI(4,5)P₂ localization temporally in live cells under both physiological and pathological conditions.

Neurons and oligodendrocytes are the building blocks of the brain. Neurons form synaptic connections and transmit signals, while oligodendrocytes, including oligodendrocyte precursor cells (OPCs) and their derivatives, are vital for central nervous system maintenance and myelination. The demand for human-specific neuron-oligodendrocyte model systems to study these interactions has grown, yet co-culture protocols remain limited. Recent advancements in the field provide methods for deriving co-cultures of neurons and OPCs from human induced pluripotent stem cells (hiPSC), each with distinct benefits and challenges. This study presents a time-efficient, reproducible method to derive neurons and O4-expressing oligodendrocytes, followed by a straightforward co-culture system that minimizes astrocyte differentiation and ensures robust neuron and oligodendrocyte populations.

Calcium-permeable AMPA receptors (CP-AMPARs) and kainate receptors (CP-KARs) play crucial roles in synaptic plasticity and are implicated in various neurological processes. Current methods for identifying neurons expressing these receptors, such as electrophysiological recordings and immunostaining, have limitations in throughput or inability to distinguish functional receptors. This protocol describes a novel approach for the vital identification of neurons containing CP-AMPARs and CP-KARs using calcium imaging. The method involves loading neurons with Fura-2 AM, a calcium-sensitive fluorescent probe, KCl application to identify all neurons, and further addition of specific AMPAR agonists (e.g., 5-fluorowillardiine) in the presence of voltage-gated calcium channel blockers and NMDAR/KAR antagonists to identify CP-AMPAR-containing neurons. CP-KAR-containing neurons are identified using domoic acid applications in the presence and absence of NASPM (a CP-AMPAR antagonist). This technique offers several advantages over existing methods, including the ability to assess large neuronal populations simultaneously, distinguish between different receptor types, and provide functional information about CP-AMPAR and CP-KAR expression in living neurons, making it a valuable tool for studying synaptic plasticity and neurological disorders.

Protein carbonylation has been known as the major form of irreversible protein modifications and is also widely used as an indicator of oxidative stress in the biological environment. In the presence of oxidative stress, biological systems tend to produce large amounts of carbonyl moieties; these carbonyl groups do not have particular UV-Vis and fluorescence spectroscopic characteristics that we can differentiate, observe, and detect. Thus, their detection and quantification can only be performed using specific chemical probes. Commercially available fluorescent probes to detect specific carbonylation in biological systems have been used, but their chemical portfolio is still very limited. This protocol outlines the methods and procedures employed to synthesize a probe, (E,Z)-2-(2-(2-hydroxybenzylidene)hydrazonyl)-5-nitrophenol (2Hzin5NP), and assess its impact on carbonylation in human cells. The synthesis involves several steps, including the preparation of its hydrazone compounds mimicking cell carbonyls, 2-Hydrazinyl 5-nitrophenol, (E,Z)-2-(2-ethylidenehydrazonyl)-5-nitrophenol, and the final product (E,Z)-2-(2-(2-hydroxybenzylidene)hydrazonyl)-5-nitrophenol. The evaluation of fluorescence quantum yield and subsequent cell culture experiments are detailed for the investigation of 2Hzin5NP effects on cell proliferation and carbonylation.

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).

Fluorescence microscopy has been widely accessible and indispensable in cell biology research. This technique enables researchers to label targets, ranging from individual entities to multiple groups, with fluorescent markers. It offers precise determinations of localization, size, and shape, along with accurate quantifications of fluorescence signal intensities. Furthermore, an ideal fluorescence microscope can achieve approximately 250 nm in lateral and 600 nm in axial resolution. Despite its integral role in these measurements, the calibration of fluorescence microscopes is often overlooked. This protocol introduces the use of 3D-Speckler (3D fluorescence speckle analyzer), a semi-automated software tool we have recently developed, for calibrating fluorescence microscopy. Calibration of fluorescence microscopy includes determining resolution limits, validating accuracy in size measurements, evaluating illumination flatness, and determining chromatic aberrations. 3D-Speckler is user-friendly and enables precise quantification of fluorescence puncta, including nanoscale 2D/3D particle size, precise locations, and intensity information. By utilizing multispectral fluorescence beads of known sizes alongside 3D-Speckler, the software can effectively calibrate imaging systems. We emphasize the importance of routine calibration for imaging systems to maintain their integrity and reproducibility, ensuring accurate quantification. This protocol provides a detailed step-by-step guide on using 3D-Speckler to calibrate imaging systems.

Overexpression of proteins in transiently transfected cells is a simple way to study basic transport mechanisms and the underlying protein–protein interactions. While expression systems have obvious drawbacks compared to in vivo experiments, they allow a quick assessment of more conserved functions, for instance, ER export or sorting of proteins in the Golgi. In a previous study, our group described the formation of ER-derived removal vesicles for the gap junction protein Cx36 in transfected HEK293T cells. These removal vesicles, termed “whorls” because of their concentric structure, were formed by Cx36 channels that failed to escape the ER. In this article, we describe an imaging protocol that can be used to determine these ER retention defects for Cx36 expressed in cultured cells. The protocol we provide here employs regular confocal microscopy, which allows for sufficient resolution to reveal the characteristic shape of ER whorls.

Lipid nanoparticle (LNP)-based drug delivery systems (DDSs) are widely recognized for their ability to enhance efficient and precise delivery of therapeutic agents, including nucleic acids like DNA and mRNA. Despite this acknowledgment, there is a notable knowledge gap regarding the systemic biodistribution and organ accumulation of these nanoparticles. The ability to track LNPs in vivo is crucial for understanding their fate within biological systems. Fluorescent labeling of LNPs facilitates real-time tracking, quantification, and visualization of their behavior within biological systems, providing valuable insights into biodistribution, cellular uptake, and the optimization of drug delivery strategies. Our prior research established reversely engineered LNPs as an exceptional mRNA delivery platform for treating ulcerative colitis. This study presents a detailed protocol for labeling interleukin-22 (IL-22) mRNA-loaded LNPs, their oral administration to mice, and visualization of DiR-labeled LNPs biodistribution in the gastrointestinal tract using IVIS spectrum. This fluorescence-based approach will assist researchers in gaining a dynamic understanding of nanoparticle fate in other models of interest.