细胞生物学

The development of patient-derived cardiac disease models has advanced rapidly due to the progress of human-induced pluripotent stem cell (hiPSC) technologies. Many protocols detail individual parts of the entire workflow, from handling hiPSCs and differentiating them into cardiomyocytes to live contraction imaging via widefield/phase-contrast and fluorescence microscopy. Here, we propose a streamlined protocol that guides users through hiPSC culture, differentiation, expansion, and functional imaging of hiPSC cardiomyocytes. First, hiPSC maintenance and handling procedures are outlined. Differentiation occurs over a two-week period, followed by selective expansion to increase the yield of hiPSC cardiomyocytes. Comprehensive characterization and quantification enable detailed contraction profiling of these cells. Designed to be low-cost, this protocol is suited for applications in drug discovery, screening, and clinical testing of patient-specific phenotypes. The addition of cardiomyocyte expansion and automated analysis distinguishes our protocol from current approaches.

Local mRNA translation in axons is crucial for the maintenance of neuronal function and homeostasis, particularly in processes such as axon guidance and synaptic plasticity, due to the long distance from axon terminals to the soma. Recent studies have shown that RNA granules can hitchhike on the surface of motile lysosomal vesicles, facilitating their transport within the axon. Accordingly, disruption of lysosomal vesicle trafficking in the axon, achieved by knocking out the lysosome–kinesin adaptor BLOC-one-related complex (BORC), decreases the levels of a subset of mRNAs in the axon. This depletion impairs the local translation of mitochondrial and ribosomal proteins, leading to mitochondrial dysfunction and axonal degeneration. Various techniques have been developed to visualize translation in cells, including translating RNA imaging by coat protein knock-off (TRICK), SunTag, and metabolic labeling using the fluorescent non-canonical amino acid tagging (FUNCAT) systems. Here, we describe a sensitive technique to detect newly synthesized proteins at subcellular resolution, the puromycin proximity ligation assay (Puro-PLA). Puromycin, a tRNA analog, incorporates into nascent polypeptide chains and can be detected with an anti-puromycin antibody. Coupling this method with the proximity ligation assay (PLA) allows for precise visualization of newly synthesized target proteins. In this article, we describe a step-by-step protocol for performing Puro-PLA in human induced pluripotent stem cell (iPSC)-derived neuronal cultures (i3Neurons), offering a powerful tool to study local protein synthesis in the axon. This tool can also be applied to rodent neurons in primary culture, enabling the investigation of axonal protein synthesis across species and disease models.

The reduction in intracellular neuronal chloride concentration is a crucial event during neurodevelopment that shifts GABAergic signaling from depolarizing to hyperpolarizing. Alterations in chloride homeostasis are implicated in numerous neurodevelopmental disorders, including autism spectrum disorder (ASD). Recent advancements in biosensor technology allow the simultaneous determination of intracellular chloride concentration of multiple neurons. Here, we describe an optimized protocol for the use of the ratiometric chloride sensor SuperClomeleon (SClm) in organotypic hippocampal slices. We record chloride levels as fluorescence responses of the SClm sensor using two-photon microscopy. We discuss how the SClm sensor can be effectively delivered to specific cell types using virus-mediated transduction and describe the calibration procedure to determine the chloride concentration from SClm sensor responses.

In nature, filamentous fungi interact with plants. These fungi are characterized by rapid growth in numerous substrates and under minimal nutrient requirements. Investigating the interaction of these fungi with their plant hosts under controlled conditions is of importance for many researchers aiming to proceed with molecular or microscopical investigations of their favorite plant–fungus interaction system. The speed of growth of these fungi complicates transferring plant–fungal interaction systems in laboratory conditions. The issue is more complicated when monoxenic conditions are desired, to ensure that only two members (a fungus and a plant) are present in the system under study. Here, two simple closed systems for investigating plant–filamentous fungi associations under laboratory, monoxenic conditions are described, along with their limitations. The plant and fungal growth conditions, methods for sampling, staining, sectioning, and subsequent microscopical imaging of colonized plant tissues with affordable, common laboratory tools are described.

Bone repair is a complex regenerative process relying on skeletal stem/progenitor cells (SSPCs) recruited predominantly from the periosteum. Activation and differentiation of periosteal SSPCs occur in a heterogeneous environment, raising the need for single cell/nucleus transcriptomics to decipher the response of the periosteum to injury. Enzymatic cell dissociation can induce a stress response affecting the transcriptome and lead to overrepresentation of certain cell types (i.e., immune and endothelial cells) and low coverage of other cell types of interest. To counteract these limitations, we optimized a protocol to isolate nuclei directly from the intact periosteum and from the fracture callus to perform single-nucleus RNA sequencing. This protocol is adapted for fresh murine periosteum, fracture callus, and frozen human periosteum. Nuclei are isolated using mechanical extraction combined with fluorescence-based nuclei sorting to obtain high-quality nucleus suspensions. This protocol allows the capture of the full diversity of cell types in the periosteum and fracture environment to better reflect the in vivo tissue composition.

Microglial cells are crucial patrolling immune cells in the brain and pivotal contributors to neuroinflammation during pathogenic or degenerative stress. Microglia exhibit a heterogeneous "dendrite-like" dense morphology that is subject to change depending on inflammatory status. Understanding the association between microglial morphology, reactivity, and neuropathology is key to informing treatment design in diverse neurodegenerative conditions from inherited encephalopathies to traumatic brain injuries. However, existing protocols for microglial morphology analyses lack standardization and are too complex and time-consuming for widescale adoption. Here, we describe a customized pipeline to quantitatively assess intricate microglial architecture in three dimensions under various conditions. This user-friendly workflow, comprising standard immunofluorescence staining, built-in functions of standard microscopy image analysis software, and custom Python scripts for data analysis, allows the measurement of important morphological parameters such as soma and dendrite volumes and branching levels for users of all skill levels. Overall, this protocol aims to simplify the quantification of the continuum of microglial pathogenic morphologies in biological and pharmacological studies, toward standardization of microglial morphometrics and improved inter-study comparability.

Communication between motor neurons and muscles is established by specialized synaptic connections known as neuromuscular junctions (NMJs). Altered morphology or numbers of NMJs in the developing muscles can indicate a disease phenotype. The distribution and count of NMJs have been studied in the context of several developmental disorders in different model organisms, including zebrafish. While most of these studies involved manual counting of NMJs, a few of them employed image analysis software for automated quantification. However, these studies were primarily restricted to the trunk musculature of zebrafish. These trunk muscles have a simple and reiterated anatomy, but the cranial musculoskeletal system is much more complex. Here, we describe a stepwise protocol for the visualization and quantification of NMJs in the ventral cranial muscles of zebrafish larvae. We have used a combination of existing ImageJ plugins to develop this methodology, aiming for reproducibility and precision. The protocol allows us to analyze a specific set of cranial muscles by choosing an area of interest. Using background subtraction, pixel intensity thresholding, and watershed algorithm, the images are segmented. The binary images are then used for NMJ quantification using the Analyze Particles tool. This protocol is cost-effective because, unlike other licensed image analyzers, ImageJ is open-source and available free of cost.

Gap junctions are transmembrane protein channels that enable the exchange of small molecules such as ions, second messengers, and metabolites between adjacent cells. Gap junctions are found in various mammalian organs, including skin, endothelium, liver, pancreas, muscle, and central nervous system (CNS). In the CNS, they mediate coupling between neural cells including glial cells, and the resulting panglial networks are vital for brain homeostasis. Tracers of sufficiently small molecular mass can diffuse across gap junctions and are used to visualize the extent of cell-to-cell coupling in situ by delivering them to a single cell through sharp electrodes or patch-clamp micropipettes. Here, we describe a protocol for pre-labeling and identification of astrocytes in acute mouse forebrain slices using Sulforhodamine 101 (SR101). Fluorescent cells can then be targeted for whole-cell patch-clamp, which allows for further confirmation of astroglial identity by assessing their electrophysiological properties, as well as for passive dialysis with a tracer such as biocytin. Slices can then be subjected to chemical fixation and immunostaining to detect dye-coupled networks. This protocol provides a method for the identification of astrocytes in live tissue through SR101 labeling. Alternatively, transgenic reporter mice can also be used to identify astrocytes. While we illustrate the use of this protocol for the study of glial networks in the mouse brain, the general principles are applicable to other species, tissues, and cell types.

Time-lapse fluorescence microscopy is a relevant technique to visualize biological events in living samples. Maintaining cell survival by limiting light-induced cellular stress is challenging and requires protocol development and image acquisition optimization. Here, we provide a guide by considering the quartet sample, probe, instrument, and image processing to obtain appropriate resolutions and information for live cell fluorescence imaging. The pleural mesothelial cell line H28, an adherent cell line that is easy to seed, was used to develop innovative advanced light microscopy strategies. The chosen red and near-infrared probes, capable of passively penetrating through the cell plasma membrane, are particularly suitable because their stimulation from 600 to 800 nm induces less cytotoxicity. The labeling protocol describes the concentration, time, and incubation conditions of the probes and associated adjustments for multi-labeling. To limit phototoxicity, acquisition parameters in advanced confocal laser scanning microscopy with a white laser are determined. Light power must be adjusted and minimized at red wavelengths for reduced irradiance (including a 775 nm depletion laser for STED nanoscopy), in simultaneous mode with hybrid detectors and combined with the fast FLIM module. These excellent conditions allow us to follow cellular and intracellular dynamics for a few minutes to several hours while maintaining good spatial and temporal resolutions. Lifetime analysis in lifetime imaging (modification of the lifetime depending on environmental conditions), lifetime dye unmixing (separation with respect to the lifetime value for the spectrally closed dye), and lifetime denoising (improvement of image quality) provide flexibility for multiplexing experiments.

In response to DNA-damaging physical or chemical agents, the DNA damage repair (DDR) pathway is activated in eukaryotic cells. In the radiobiology field, it is important to assess the DNA damage effect of a certain irradiation regime on cancer cells and compare it to the effect on non-transformed cells exposed to identical conditions. The first step in the DNA repair mechanism consists of the attachment of proteins such as the phosphorylated histone γ-H2AX (p-γ-H2AX) to DNA double-strand breaks (DSB) in the nucleus, which leads to the formation of repairing foci. Therefore, imaging methods were established to evaluate the presence of foci inside the nucleus after exposure to DNA-damaging agents. This approach is superior in sensitivity to other methods, such as the comet assay or the pulsed-field gel electrophoresis (PFGE), that allow direct detection of cleaved DNA fragments. These electrophoresis-based methods require high ionizing radiation dosages and are difficult to reproduce compared to imaging-based assays. Conventionally, the number of foci is determined visually, with limited accuracy and throughput. Here, by exploring the effect of laser-plasma accelerated electrons FLASH irradiation on cancer cells, we describe an image cytometry protocol for the quantification of foci with increased throughput, upon large areas, with increased precision and sample-to-sample consistency. It consists of the automatic scanning of fluorescently labeled cells and using a gating strategy similar to flow cytometry to discriminate cells in co-culture based on nuclei elongation properties, followed by automatic quantification of foci number and statistical analysis. The protocol can be used to monitor the kinetics of DNA repair by quantification of p-γ-H2AX at different time points post-exposure or by quantification of other DNA repair proteins that form foci at the DNA DSB sites. Also, the protocol can be used for quantifying the response to chemical agents targeting DNA. This protocol can be performed on any type of cancer cells, and our gating strategy to discriminate cells in co-culture can also be used in other research applications.