β1-integrin is a transmembrane glycoprotein that heterodimerizes with multiple α subunits and functions as a key receptor for extracellular matrix proteins. It contains a large extracellular ligand-binding domain, a single-pass transmembrane region, and a short cytoplasmic tail involved in intracellular signaling that supports cell adhesion, migration, and tissue development. Fluorescent β1-integrin fusion proteins are challenging to image in standard cell lines because endogenous β1-integrin competes with the fusion protein for maturation and α-subunit pairing, often resulting in aggregation or endoplasmic reticulum retention, and the consequent perinuclear fluorescence. This article describes a straightforward transient expression strategy that yields properly processed β1-integrin-ECFP at the plasma membrane in murine HC11 cells. The method uses a bicistronic construct that expresses β1-integrin-ECFP while simultaneously reducing endogenous Itgb1 via a short hairpin RNA targeting its 3' untranslated region, thereby sparing the recombinant transcript. This approach enables robust membrane localization of the fusion protein and enrichment at structures consistent with focal adhesions. © 2026 Wiley Periodicals LLC. Basic Protocol: Construction and transient expression of a bicistronic β1-integrin-ECFP/shItgb1 plasmid to achieve plasma membrane targeting in HC11 cells Support Protocol 1: Glass coverslip cleaning and surface functionalization for cell adhesion Support Protocol 2: Cell fixation using paraformaldehyde.

Quantitative RNA imaging in large plant tissues has historically been challenging because of limited spatial resolution, low signal-to-noise ratios, and the largely qualitative nature of traditional RNA in situ hybridization methods. Hybridization chain reaction-RNA fluorescence in situ hybridization (HCR RNA-FISH) coupled with high-resolution microscopy enables sensitive detection of RNA molecules at cellular resolution. However, quantitative approaches that combine improved tissue accessibility with robust computational pipelines for single-cell transcript quantification in plants remain limited. Here, we present a quantitative HCR RNA-FISH protocol for the developing maize inflorescence. We describe a 4-day workflow that includes fixation, agarose immobilization, vibratome sectioning, probe hybridization, amplification, and mounting and enables multiplexed detection of transcripts at the cellular level in maize ear and tassel primordia. In addition, we provide a Python-based image analysis pipeline for (i) cell segmentation, (ii) RNA spot quantification, (iii) assignment of spots to cells, and (iv) data representation. The scripts can be easily run on Jupyter notebooks and are available on GitHub. Overall, this protocol highlights the importance of integrating robust imaging strategies with quantitative and reproducible data analysis frameworks to extract biologically meaningful insights from imaging data. © 2026 Wiley Periodicals LLC. Basic Protocol: Quantitative RNA imaging in sections of maize ear and tassel primordia using HCR RNA-FISH.

Learning and memory are essential for the acquisition, storage, and recall of information, enabling organisms to adapt to changes in their environment. Associative learning begins when a stimulus, either positive or negative in valence, is presented alongside a neutral stimulus. This drives the development of an association between the stimuli, leading to the subsequent establishment of a learned behavioral response to the previously neutral stimulus. Associative learning is well conserved across species, from invertebrates to humans, and is highly amenable to experimental manipulation. Drosophila melanogaster is a valuable model for dissecting the mechanisms underlying associative learning and memory, given its genetic tractability, low cost, and the high‐throughput nature of fly behavioral assays. Here, we describe two distinct associative learning paradigms in the Drosophila model that employ olfaction to establish short‐term learned associations using a Y‐maze assay. We first describe a novel negative associative learning paradigm employing a mechanical aversive stimulus. We next describe a positive associative learning paradigm in which flies learn to approach an odor paired with an appetitive sucrose reward. Overall, these protocols offer an inexpensive and streamlined approach to investigating associative learning under a multitude of different contexts. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Negative associative learning paradigm utilizing mechanical stimulationBasic Protocol 2: Positive associative learning paradigm utilizing sucrose feeding

For several years, scientists have focused on studying mesenchymal stem cells because of their ability for self-regeneration, their potential for differentiation into multiple lineages (e.g., osteogenic, chondrogenic, and adipogenic cells), and their low immunogenicity and remarkable capacity to modulate the immune system. The importance of these cells ranges from preserving tissue health and repairing injured tissues in their nearby areas, to their use in scientific investigation for the treatment of neurodegenerative, autoimmune, and cardiovascular diseases. Despite the availability of various tissue sources, such as bone marrow and adipose tissue, their collection and handling may not always be easily achievable. However, dental tissues, such as the pulp and periodontal ligament, are relatively accessible supplies that do not require complex or stressful interventions for the patient. Here, we provide a detailed description of each step involved in the isolation and characterization of mesenchymal stem cells from the pulp and periodontal ligament using monoclonal antibodies ensuring a high level of effectiveness. In addition, we also describe a technique to generate the cellular presenescence state. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Isolation of human periodontal ligament and dental pulp stem cells Support Protocol 1: Characterization of human periodontal ligament and dental pulp stem cells Support Protocol 2: Induction of presenescence of human periodontal ligament and dental pulp stem cells.

Exosomes are small extracellular vesicles (50 to 150 nm in size) that carry different kind of biomolecules in their lumen and facilitate intercellular communication. Serum contains a large number of exosomes (∼1012/ml of serum) that contain relatively more small RNAs than large RNAs. Quantifying the absolute number of specific miRNA molecules in exosomes is critical for understanding their functional relevance and developing them for diagnostic and therapeutic applications. This article describes an exosome copy number assay that combines nanoparticle tracking analysis (NTA) to quantify vesicle concentration with absolute quantification of miRNA using a standard curve-based qPCR approach. The workflow includes optimized procedures for vesicle isolation, particle measurement, RNA extraction, and absolute quantification, enabling calculation of miRNA copy number per exosome. The method can be used to generate reproducible and accurate results across diverse biological samples. By offering precise molecular quantification, these protocols provide a practical framework to advance exosome biology and support the development of exosome-based biomarkers and therapies. © 2026 Wiley Periodicals LLC. Basic Protocol 1: Isolation of exosomes from human serum by ultracentrifugation Support Protocol: Exogenous spike-in controls and troubleshooting Basic Protocol 2: NTA for exosome enumeration Basic Protocol 3: Extraction of RNA from purified exosomes from serum Basic Protocol 4: Reverse transcription (cDNA synthesis and preamplification) and real-time PCR setup Basic Protocol 5: Copy number assay (standard curve preparation) Basic Protocol 6: Calculation of miRNA copy number per vesicle (absolute quantification by qPCR and NTA normalization).

The unpredictable chronic stress (UCS) protocol, originating in the 1980s, has contributed to depression research by inducing behavioral and physiological changes in rodents that resemble the symptoms observed in patients. Its translational potential led to widespread adoption, but adaptations and variations to the protocol have raised concerns about reproducibility. Over a decade ago, our laboratory adapted the UCS protocol for zebrafish (Danio rerio), aiming to bridge species-specific gaps in stress research. Since then, several studies have reported alterations in outcomes related to the clinical manifestations of stress-related disorders, providing evidence on the applicability of the model for testing drugs and other therapeutic strategies. However, there is substantial heterogeneity in protocols and research findings, hindering comparability across studies. To address these challenges, we describe a 14-day UCS protocol for adult zebrafish in detail, involving a series of varied stressors administered twice daily. This comprehensive protocol includes stressors, such as tank changes, net chasing, overcrowding, low water levels, and temperature fluctuations. Each step is designed to ensure reproducibility and to mimic the unpredictable and varied nature of stress experienced by humans. By establishing standardized procedures, we aim to enhance the consistency, reliability, and translational potential of UCS research using zebrafish, ultimately contributing to more robust and comparable findings across studies. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: UCS protocol for adult zebrafish.

Cell Painting (CP) methods use a combination of fluorescent dyes to label multiple cellular compartments simultaneously, enabling the comprehensive analysis of phenotypic changes through morphological profiling. Here, we present a detailed protocol for the Cell Painting PLUS (CPP) method along with an automated image and data analysis strategy. In CPP, the iterative staining, elution, and re-staining of cells with seven fluorescent dyes enable multiplexed analysis of nine cellular compartments and organelles. Each dye is captured in individual imaging channels to specifically distinguish effects on the plasma membrane, actin cytoskeleton, cytoplasmic and nucleolar RNA, lysosomes, nuclear DNA, endoplasmic reticulum, mitochondria, and Golgi apparatus. During the image analysis procedure, 894 morphological features (readouts) are extracted from single cells to generate comprehensive phenotypic profiles that resemble morphological perturbations. For efficient processing of the extracted features, we provide an automated data analysis workflow, which includes quality control, data normalization, and various data visualization tools. This workflow is based on a customized CPPAnalyzer Jupyter notebook and a CPPManager KNIME workflow, which can be easily applied without any special bioinformatics knowledge. In this way, CPP expands the multiplexing capacity, customizability, and, importantly, organelle specificity of the available CP-based screening methods. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Seeding of U2OS, MCF-7, or HepG-2 cells in multiwell plates and treatment of cells with compounds Support Protocol 1: Preparation of assay-ready compound plate Alternate Protocol: Seeding and differentiation of primary RPTEC-TERT1 cells in multiwell plates Basic Protocol 2: Staining and imaging of cells using Cell Painting PLUS Basic Protocol 3: Image analysis Basic Protocol 4: Data normalization and data quality control Basic Protocol 5: Visualization of data using CPPManager KNIME workflow.

Effective evaluation and assessment of research-focused programs, including centers, core facilities, and institutes, is crucial but inherently challenging. Faculty members are often departmentalized, yet their research necessitates campus-wide access to infrastructure, specialized equipment, students, and interdisciplinary collaborators, thus creating assessment and reporting challenges. Additionally, it can be challenging to demonstrate return on investment for new and emerging programs as well as those at institutions without an extensive history of research. Social network analysis (SNA) tools may be used to address these challenges. SNA provides a data-driven understanding of collaboration dynamics in programs designed to build research community and provides a method to quantify program impact and demonstrate the effectiveness of capacity-building investments in fostering collaborative research and scientific output. Additionally, SNA is useful in pinpointing influential researchers who act as central nodes or bridges, and may guide future efforts in mentorship, grant development, and recruitment, by highlighting areas of productive collaboration, or alternatively, potential fragmentation in the network. The results may be used to guide future resource allocation, inform new initiatives within the program, or alternatively, target interventions if needed to strengthen a specific research area. Initial analysis can help a program assess collaborative health of a research community by providing a baseline for tracking changes in network structure and collaboration patterns over time, setting the stage for longitudinal evaluation. This article outlines the steps required to carry out SNA of bibliometric data from a biomedical research community. The article describes the acquisition of data through PubMed, application of VOSviewer and Gephi, data visualization, statistical analysis, and generation of figures suitable for publication, including examples of results designed for specific audiences and stakeholders. The article also covers creating an author thesaurus for disambiguation of authors who publish under multiple names. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Obtaining data Basic Protocol 2: Initial bibliometric data processing and co-authorship network construction using VOSviewer Basic Protocol 3: Interfacing VOSviewer data with Gephi Basic Protocol 4: Advanced network visualization and statistical analysis in Gephi Basic Protocol 5: Creating impactful figures Basic Protocol 6: Identifying key individuals or groups of individuals within the network Support Protocol 1: VOSviewer author thesaurus file generation Support Protocol 2: Quick-start protocol: SNA for program evaluation.

Chimeric antigen receptor (CAR)-T cell treatment has shifted the paradigm of cancer immunotherapy, especially in B-cell malignancies, with around an 80% response rate in acute lymphoblastic leukemia. Emerging data on the use of CAR-T cells in the treatment of autoimmune diseases, such as lupus, indicate another significant advance in this therapeutic field and highlight its promising potential for the future. Despite its remarkable therapeutic potential, the manufacturing of CAR-T cells, including the production of critical components such as high-titer lentiviral vector (LV), remains a significant challenge, limiting the broader accessibility and the ease of manufacturing and administrationof this therapy. Lack of optimized and harmonized protocols makes the manufacturing of CAR-T cells cumbersome. To ease some of the challenges of CAR-T cell manufacturing, we optimized LV production, with an emphasis on enhancing viral titers using different transfer-to-helper plasmid ratios, optimizing transfection efficiency in HEK293T cells, and fine-tuning peripheral blood mononuclear cell activation and transduction conditions. We present a set of optimized protocols for the scalable production of third-generation LVs for CAR-T cell generation. This approach facilitates preclinical and translational research by providing a framework for the reproducible, economical, and effective production of CAR-T cells. © 2026 Wiley Periodicals LLC. Basic Protocol 1: Plasmid ratio screening Basic Protocol 2: Viral concentration via ultracentrifugation Basic Protocol 3: FACS-based titration assay Basic Protocol 4: Peripheral blood mononuclear cell (PBMC) isolation Basic Protocol 5: PBMC activation for effective transduction Basic Protocol 6: CAR-T cell generation with lentivirus Support Protocol: Flow cytometry gating strategies.