The airway epithelium interfaces with the external environment through its apical surface and with the extracellular matrix (ECM) through its basolateral surface. To model this organization in vitro, we developed a decellularized ECM-incorporated apical-out airway organoid (dECM-AoAO) platform in which human bronchial epithelial cells (HBECs) self-assemble around human lung-derived decellularized ECM microparticles (dECM-MPs). This configuration preserves apical-out polarity while enabling direct epithelial-ECM interactions. Here, we describe a protocol for the vacuum filtration and quantification of dECM-MPs, the generation of dECM-AoAOs, and ultimately, whole-mount immunofluorescence staining for organoid characterization. Key features • This protocol incorporates dECM as size-refined microparticles, enabling a consistent and reproducible ECM input. • The workflow uses suspension culture to generate apicalout airway organoids, eliminating the need for matrix embedding while preserving ECM-cell interactions. • dECM-MPs are directly wrapped by epithelial cells, simplifying handling and supporting native-like epithelial-ECM integration. • The system is compatible with wholemount immunofluorescence staining for comprehensive epithelial lineage analysis.
Chemotherapy-resistant persister cells are a major driver of cancer recurrence, yet their epigenetic basis remains poorly characterized. This protocol describes a computational pipeline for identifying DNA-binding factors (DBFs) that are enriched in accessible chromatin that collectively define a persister cell signature (PCS). Starting from single-nucleus ATAC-seq (snATAC-seq) data processed through the 10x Genomics CellRanger ARC pipeline, this protocol covers (1) the creation of a Seurat/Signac object with ATAC peaks, (2) the optional integration of DNA-binding data from the ReMap2022 database as a per-cell chromatin module assay, (3) differential accessibility analysis across clinically defined comparison groups, and (4) identifying and defining the top enriched DBFs as the PCS. This approach is applicable to any snATAC-seq dataset in which cells can be grouped by clinical response, treatment status, or resistance phenotype. Key features • Integrated analysis of chromatin accessibility data with publicly available DNA-binding data is a useful technique to identify potential epigenetic biomarkers. • Single-nucleus ATAC-seq from clinically relevant samples can be used to identify binding enrichment of specific DNA-binding factors in sub-populations of cells. • This protocol was used to identify a persister cell signature that was able to predict chemotherapy resistance in high-grade serous ovarian cancer.
Ammonium (NH4 +) is the primary inorganic nitrogen source for rice (Oryza sativa L.). Substantial progress has been made in characterizing the functions of ammonium transporters (AMTs) in roots; however, the regulatory dynamics governing subcellular ammonium compartmentation after its entry into cells, particularly its vacuolar sequestration and efflux back to the external environment, remain poorly understood. This knowledge gap stems mainly from two factors: the difficulty of applying conventional detection methods at the organellar scale and interference caused by nonspecific ion adsorption to the cell wall of intact roots. To address these challenges, we present a detailed and reproducible protocol for real-time measurement of net NH4 + fluxes in rice roots, root protoplasts, and isolated vacuoles using non-invasive micro-test technology (NMT). The protocol covers the preparation of protoplasts and vacuoles from rice roots, the configuration and calibration of the NMT system, and the step-by-step measurement of net NH4 + fluxes at three distinct biological levels (intact roots, protoplasts, and vacuoles). By employing a unified sample preparation and measurement strategy, this protocol enables quantification of net uptake fluxes across the plasma membrane, characterization of net efflux dynamics under specific conditions, and indirect estimation of vacuolar sequestration capacity using the isolated vacuole system. Overall, this protocol provides a flexible and robust framework for studying NH4 + homeostasis in plants and is readily adaptable to different crop species, treatment conditions, and experimental objectives. Owing to its modular design and compatibility with standard NMT equipment, it can be readily adopted by laboratories seeking to investigate nitrogen transport mechanisms in plants. Key features • Allows for testing of NH4 + fluxes in roots, protoplasts, and vacuoles. • Applicable to plants grown under different culture systems, including Arabidopsis thaliana grown in dishes and rice grown in hydroponic systems. • Supports both long-term and transient stress treatments. • Real-time monitoring.
Expressing large DNA constructs in the native three-dimensional brain microenvironment remains technically challenging. Although viral vectors provide high transduction efficiency and cell-type selectivity, their genetic payload capacity is limited. Various non-viral approaches have been used in brain tissue, but they may compromise tissue viability or require specialised equipment, such as biolistic delivery or electroporation. We present an adapted protocol for delivering the large DNA vector encoding the optical PIEZO1 sensor GenEPi into brain tissue to enable sensor expression in pyramidal neurons. By applying DNA-Lipofectamine liposomes directly to the slice surface, we achieved efficient, minimally invasive transfection of pyramidal neurons in the CA1 and CA3 regions of organotypic hippocampal slices. PIEZO1 sensor expression was detectable as early as 7 days after transfection, increased with longer tissue maintenance, and was sustained for 3-4 weeks in vitro. This protocol describes a cost-effective, non-invasive approach that preserves cell viability and enables investigation of PIEZO1-mediated mechanotransduction in a native brain microenvironment. Key features • DNA-Lipofectamine liposomes are applied directly to slice surface, enabling efficient transfection of superficial hippocampal neurons, important for imaging experiments performed using upright microscope systems. • The protocol provides a cost-effective gene delivery approach that requires only small volumes of DNA and transfection reagent. • Robust expression of the PIEZO1 sensor GenEPi is achieved within a relatively short time (approximately 1 week after transfection). • The method is compatible with long-term tissue maintenance, with neuronal viability and GenEPi expression maintained for up to 3-4 weeks after transfection.
Small ubiquitin-like modification (SUMOylation) is a crucial post-translational modification that modulates protein stability, localization, and interaction dynamics. Despite the identification of thousands of putative small ubiquitin-like modifier (SUMO) substrates, functional validation remains challenging due to the low abundance and highly dynamic nature of SUMOylated proteins. Here, we present a protocol for detecting protein SUMOylation, integrating bioinformatic site prediction, and rapid substrate screening via in vivo tobacco transient expression and in vitro E. coli assay, followed by precise validation using transgenic Arabidopsis lines. However, detection of low-abundance SUMOylated proteins may require coupling with mass spectrometry, and the in vitro system does not fully recapitulate the complex regulatory network in vivo. This workflow provides a useful tool for studying SUMOylation in plants. Key features • Integrates bioinformatic prediction, in vitro validation, and in vivo confirmation for SUMOylation analysis. • E. coli co-expression system enables rapid SUMOylation detection without protein purification. • Arabidopsis transgenic line system confirms SUMOylation under physiological conditions. • The protocol is applicable to most Arabidopsis proteins.
Surface receptor engagement governs T-cell activation. Since these surface receptors undergo extensive glycosylation, lectin-mediated crosslinking of these glycosylated surface receptors has the potential to modulate signaling. Here, we systematically evaluate the abilities of recombinant human galectins in triggering immune responses. We describe how to apply the human galectins to modulate Jurkat E6-1 cell activation by measuring the expression level of cellular surface CD69 and the mRNA of IL-2. To validate the protocol, we confirmed that galectin-3 and galectin-8 variants 1 and 2 reproducibly induce CD69 and IL-2 expression on Jurkat E6-1 cells. Our approach offers a galectin-based toolset to study how glycosylation modulates human adaptive immunity. Key features • Systematic screening of the recombinant human galectin 1, 3, 7, 8 (both of variant 1 and variant 2) for Jurkat E6-1 cell modulatory activity. • Mechanism-based approach utilizing glycan crosslinking to induce receptor engagement. • Standardized workflow to evaluate extracellular galectin-driven immune activation.
Progressive neurodegeneration linked to the accumulation of misfolded proteins is a hallmark of several neurodegenerative disorders, including Parkinson's disease, Huntington's disease, and Alzheimer's disease. Dysfunction in the protein homeostasis machinery correlates with pathology. The chaperone protein DNAJB6 is expressed in neurons and oligodendrocytes and has been shown to play a key role in preventing amyloid aggregation by binding to amyloidogenic proteins and facilitating their refolding or degradation, in cooperation with other chaperones. Here, we describe a simple and feasible assay that enables high-throughput screening for DNAJB6 activity in a plate reader format. We use genetically engineered HEK293 cells that stably express DNAJB6 fused to either CFP or YFP. These cells can be plated into multi-well plates, and the fluorescence resonance energy transfer (FRET) signal can be measured for analysis of DNAJB6 dimerization, which is linked to DNAJB6 activity. The protocol can be used for drug screening and to identify compounds that increase DNAJB6 dimerization, and can serve as a starting point for finding new medicines that act through modulating DNAJB6 activity. Key features • The protocol requires a plate reader capable of FRET analysis and bandwidth adjustment for CFP/YFP separation. It was developed using a CLARIOstar plate reader. • The protocol requires access to the authors' FRET DNAJB6 cell line or equivalent cells with stable expression of CFP/YFP-DNAJB6. • The assay measures DNAJB6 dimerization and can potentially be adapted to other proteins whose functional state is linked to dimerization activity. • The protocol is useful for compound screening purposes and requires pre-existing knowledge of basic cell culture.
Super-resolution fluorescence microscopy enables the visualization of protein structures at nanometer resolution, providing insights into receptor organization on the plasma membrane that are essential for the development and optimization of immunotherapies. In this context, monoclonal antibodies are employed, which typically bind only a subset of available membrane receptors, due to steric hindrance or otherwise limited epitope accessibility, to quantify the accessible targets. These accessible targets, rather than the total receptor density, are critical for determining therapeutic efficacy. Here, we present a simplified, robust protocol to quantify antibody-accessible endogenous receptors using monoclonal antibodies directly labeled with fluorescent dyes in combination with total internal reflection fluorescence (TIRF) direct stochastic optical reconstruction microscopy (dSTORM). The method employs optimized labeling and fixation conditions to preserve the native receptor distribution, enabling precise quantification of accessible receptors and their stoichiometry at single-molecule resolution. Omitting secondary antibodies and minimizing fixation-induced artifacts prevents artificial clustering and maintains the physiological binding pattern of therapeutic antibodies. The standardized workflow delivers therapy-relevant information about receptor accessibility and organization underlying therapeutic antibody binding, thereby advancing the mechanistic understanding of immunotherapy resistance and personalized treatment strategies across diverse membrane protein targets. Key features • Influence of fixation conditions on receptor epitope accessibility. • Use of therapeutic antibodies for quantitative estimation of receptor availability relevant to immunotherapy. • Fluorophore localizations provide information on antibody binding events below the optical resolution limit.
Super-resolution microscopy has transformed our ability to visualize subcellular structures, but its application in plant biology remains challenging due to the optical complexity of plant tissues. Here, we present a detailed protocol for tau-STED microscopy (Leica Microsystems), which combines stimulated emission depletion (STED) with fluorescence lifetime imaging (FLIM) to achieve nanoscale resolution while minimizing phototoxicity. This method leverages time-correlated single-photon counting (TCSPC) to separate fluorescence signals based on their lifetimes, enhancing signal specificity and enabling the visualization of elusive subcellular compartments in Arabidopsis thaliana root tips. The protocol covers sample preparation, fluorophore selection, microscope configuration, image acquisition, and data analysis, providing a step-by-step guide to optimize tau-STED imaging for plant cell biology. By addressing the unique challenges of plant tissue imaging, such as autofluorescence, refractive index mismatches, and light scattering, this approach facilitates super-resolution imaging of intracellular structures, including the plant endoplasmic reticulum-Golgi intermediate compartment (ERGIC). This protocol is designed to be accessible to researchers with basic microscopy experience and offers a robust framework for exploring subcellular dynamics in plants with unprecedented detail. Key features • tau-STED integrates STED signals with fluorescence lifetime via phasor analysis at confocal speeds, enabling low-noise super-resolution imaging. • Morphometry analysis workflow at super resolution.
