细胞生物学

AMPA-type receptors are transported large distances to support synaptic plasticity at distal dendritic locations. Studying the motion of AMPA receptor⁺ vesicles can improve our understanding of the mechanisms that underlie learning and memory. Nevertheless, technical challenges that prevent the visualization of AMPA receptor⁺ vesicles limit our ability to study how these vesicles are trafficked. Existing methods rely on the overexpression of fluorescent protein-tagged AMPA receptors from plasmids, resulting in a saturated signal that obscures vesicles. Photobleaching must be applied to detect individual AMPA receptor⁺ vesicles, which may eliminate important vesicle populations from analysis. Here, we present a protocol to study AMPA receptor⁺ vesicles that addresses these challenges by 1) tagging AMPA receptors expressed from native loci with HaloTag and 2) employing a block-and-chase strategy with Janelia Fluor-conjugated HaloTag ligand to achieve sparse AMPA receptor labeling that obviates the need for photobleaching. After timelapse imaging is performed, AMPA receptor⁺ vesicles can be identified during image analysis, and their motion can be characterized using a single-particle tracking pipeline.

The Seahorse 96 XF Analyzer (Agilent Technologies, Santa Clara, CA, USA) has been an effective tool in non-invasively measuring mitochondrial function for the past decade. It is a high-throughput respirometer that is considered the “gold standard” for quantifying mitochondrial function and bioenergetics in cells. Peripheral blood mononuclear cells (PBMCs) play a selective role in immune system responses and are key components of human immunity. Recent studies have suggested that these cell populations provide an overview of systemic changes within the body and therefore provide a source of sensitive biomarkers. Assessing mitochondrial function in PBMCs has been shown to provide an indication of metabolic stress associated with diseases such as diabetes and neurodegenerative conditions such as Alzheimer’s disease. In this protocol, we use two adhesive compounds, Poly-D-Lysine (PDL) and Poly-L-Lysine (PLL), at 50 μg/mL each per well, to immobilize PBMCs to a specialized Seahorse microplate to perform mitochondrial stress assay using the Seahorse Analyzer. We compared six cell densities of PBMCs to identify the optimal cell density for use in Seahorse Mito Stress analysis. This protocol includes the immobilization of freshly isolated PBM cells into a Seahorse microplate, hydration and calibration of the sensor cartridge, cell seeding, running the Seahorse Analyzer for the Mito Stress test, and simple data analysis to compare the effectiveness of PLL and PDL as the coating agent for PBMCs. The data analysis indicates that there is no statistical difference between PLL and PDL.

Plastic pollution presents a looming danger to the environment and virtually all life on planet Earth. Especially pernicious are nanoplastics (NPs), which are plastic fragments with dimensions ≤1 μm. Conventional detection methods are ineffective for NPs, while their high specific surface area renders them efficient carriers of toxic substances; additionally, they may even be inherently toxic. Although NP waste chiefly arises from environmental weathering of larger plastic fragments, most published studies employed manufactured pristine NPs of uniform size and shape. Furthermore, almost all NP effects were studied using polystyrene (PS) as a convenient model material, despite PS accounting for <6% of all plastic pollution. There is thus an urgent need to expand investigations of environmental NP pollution and effects on biota. The present work provides a comprehensive roadmap for studying the effects of “real-world” NP pollution on living systems, using, for example, lung alveolar epithelial cells on which such NPs deposit by breathing ambient air. Herein, we describe detailed in-house methods to fabricate various NPs that are weathered with UV light and O₃ gas exposure to more closely mimic real environmental NPs. We also illustrate a simple and straightforward bioelectrical method for assessing passive and active ion transport properties of primary rat lung alveolar epithelial cell monolayers as a model for the distal mammalian lung exposed to one of the generated NPs. This protocol allows researchers to rapidly and more accurately assess the biological impact of various simulated environmental NPs on a vulnerable air–blood barrier in the lung.

Centrosomes are vital eukaryotic organelles involved in regulating cell adhesion, polarity, mobility, and microtubule (MT) spindle assembly during mitosis. Composed of two centrioles surrounded by the pericentriolar material (PCM), centrosomes serve as the primary microtubule-organizing centers (MTOCs) in proliferating cells. The PCM is crucial for MT nucleation and centriole biogenesis. Centrosome numbers are tightly regulated, typically duplicating once per cell cycle, during the S phase. Deregulation of centrosome components can lead to severe diseases. While traditionally viewed as stable structures, centrosomes can be inactivated or disappear in differentiating cells, such as epithelial cells, muscle cells, neurons, and oocytes. Despite advances in understanding centrosome biogenesis and function, the mechanisms maintaining mature centrosomes or centrioles, as well as the pathways regulating their inactivation or elimination, remain less explored. Studying centrosome maintenance is challenging as it requires the uncoupling of centrosome biogenesis from maintenance. Tools for acute spatial-temporal manipulation are often unavailable, and manipulating multiple components in vivo is complex and time-consuming. This study presents a protocol that decouples centrosome biogenesis from maintenance, allowing the study of critical factors and pathways involved in the maintenance of the integrity of these important cellular structures.

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.

In vitro lymphocyte proliferation assays are essential for assessing immune responses and antiproliferative drug efficacy. Such assays rely on antigen presentation or mitogen stimulation, with performance determined by reagent concentration and incubation time. Although splenocytes are often used, peripheral blood mononuclear cells (PBMCs) offer more accessible and practical sampling. However, a streamlined protocol for porcine PBMCs proliferation with robust batch analysis has been lacking. We therefore developed a detailed workflow for inducing proliferation in cryopreserved porcine PBMCs using 5 μg/mL concanavalin A (ConA). The protocol covers cell isolation, cryopreservation, ConA stimulation, CD4+ T-cell staining, flow cytometry acquisition and gating on an Attune NxT instrument, and batch analysis with FCS Express^TM 7.18. This approach yielded 78.9% viable cells, of which 33.8% were CD4+ lymphocytes. Moreover, 93.9% (n = 216) of cells proliferated, yielding up to nine cell generations. Batch analysis in FCS Express^TM enhanced the accuracy and interpretation of proliferation metrics. This validated protocol provides a reliable framework for generating consistent proliferation data in porcine immunology studies.

Amyloplasts, non-photosynthetic plastids specialized for starch synthesis and storage, proliferate in storage tissue cells of plants. To date, studies of amyloplast replication in roots and the ovule nucelli from various plant species have been performed using electron and fluorescence microscopy. However, a complete understanding of amyloplast replication remains unclear due to the absence of experimental systems capable of tracking their morphology and behavior in living cells. Recently, we demonstrated that Arabidopsis ovule integument could provide a platform for live-cell imaging of amyloplast replication. This system enables precise analysis of amyloplast number and shape, including the behavior of stroma-filled tubules (stromules), during proplastid-to-amyloplast development in post-mitotic cells. Here, we provide technical guidelines for observing and quantifying amyloplasts using conventional fluorescence microscopy in wild-type and several plastid-division mutants of Arabidopsis.

Since the establishment of the iSLK-BAC16 cell culture system, iSLK-BAC16 cells and their derivatives have been widely used for Kaposi’s sarcoma-associated herpesvirus (KSHV) studies. However, iSLK-BAC16 cells can be difficult to work with, in part due to the lack of standardized protocols and conflicting troubleshooting suggestions. Here, we describe the protocol for general iSLK-BAC16 cell culture and reactivation, which induces lytic KSHV replication and virion production. This protocol achieves robust levels of KSHV reactivation in our hands and can be readily used for studies of KSHV lytic infection mechanisms.

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.

Long-term depression (LTD), a key form of synaptic plasticity, is typically induced through regulated Ca²⁺ entry via NMDA receptors and achieved by prolonged (up to hundreds of seconds) low-frequency presynaptic stimulation or bath application of NMDA receptor agonists. Electrophysiological approach to LTD induction requires specialized equipment, while bath applications limit productivity, as only one neuron per sample may be recorded. Here, we present a simple and effective protocol for pharmacological modeling of LTD in primary cultured neurons. This approach relies on highly localized iontophoretic application of NMDA, which induces LTD in individual cells, enhancing experimental throughput. We have analyzed spatio-temporal patterns of iontophoretic drug delivery and demonstrated how this technique may be combined with electrophysiological and live-cell imaging approaches to investigate LTD-related changes in synaptic strength and Ca²⁺-dependent signaling of neuronal Ca²⁺ sensor proteins.

Continuous and balanced bone remodeling is essential for maintaining mechanical integrity, mineral homeostasis, and hematopoiesis. Dysregulated bone metabolism develops pathological conditions, such as osteoporosis and bone metastasis. Functional and analytical recapitulation of bone remodeling in vitro is critical for advancing our understanding of bone mineral metabolism, disease mechanisms, and drug development. However, conventional models fail to replicate the essential complexity of the bone extracellular matrix (ECM) and the dynamic interplay between bone-forming osteoblasts and bone-resorbing osteoclasts. Recently, we developed an osteoid-mimicking demineralized bone paper (DBP) by thin-sectioning demineralized bovine compact bone matrix. DBP supports osteoblastic mineral deposition and the subsequent transition to bone-lining cells. When co-cultured with bone marrow mononuclear cells under biochemical stimulation, osteoblasts shift their regulatory secretion profiles and effectively induce osteoclastogenesis. The semi-transparent nature of DBP, combined with primary osteogenic cells retrieved from DsRed and eGFP reporter mice, enables longitudinal fluorescent monitoring of these multicellular processes and quantitative analysis. In this protocol, we describe the methods for DBP generation, reconstituting mineralized bone tissue complexity with osteoblasts, and recapitulating the bone remodeling cycle through bone marrow monocytes co-culture under biochemical stimulation, offering a useful platform for the related and broader research community.