STAR Protocols

Protocol for preparing and characterizing samples for combined microsecond freeze-hyperquenching and electron paramagnetic resonance spectroscopy #protocol #starprotocols #cellpress

Computational protocol for hierarchical Bayesian modeling of perception and generalization in fear conditioning #protocol #starprotocols #cellpress

Protocol for efficient CRISPRi-mediated silencing of retrotransposons in human pluripotent stem cells #protocol #starprotocols #cellpress

Protocol for generating megakaryocytes from patient induced pluripotent stem cells for disease modeling and compound screening #protocol #starprotocols #cellpress

Protocol for formulation and evaluation of phytosterol-based lipid nanoparticles as cholesterol alternatives for mRNA delivery #protocol #starprotocols #cellpress

Protocol for the preparation of flexible material from the mycelium of wood-decay fungal strains and mechanical property investigation #protocol #starprotocols #cellpress

Protocol for evaluating seedling greening capacity during dark-to-light transition #protocol #starprotocols #cellpress

Protocol for purifying recombinant HBV capsids from HepG2 cells and comparative phosphorylation analysis by phosphate-affinity SDS-PAGE #protocol #starprotocols #cellpress

Protocol for the generation of a human-derived nasal epithelial model and induction of tissue-resident memory-like T cells in a co-culture system #protocol #starprotocols #cellpress

Protocol for automated analysis of biological images using Python code #protocol #starprotocols #cellpress

Protocol for generating an in vitro 3D multicellular culture model of ovarian high-grade serous carcinoma The development of translational ovarian cancer models to investigate and overcome treatment resistance, while accounting for the impact of the tumor microenvironment, is critical. Here, we present a protocol to establish a multicellular culture model that retains both genetic complexity and the microenvironment of patient tumors and is amenable to molecular and phenotypic analyses and high-throughput drug testing. We describe steps for culturing and characterizing stromal cells derived from cry...

Protocol for generating an in vitro 3D multicellular culture model of ovarian high-grade serous carcinoma #protocol #starprotocols #cellpress

Protocol for KAT8 conditional knockout mice generation and its application in 4NQO-induced esophageal tumor model 4-Nitroquinoline 1-oxide (4NQO), a quinoline derivative, induces tumor formation in mice following a multi-stage pattern analogous to human tumors, mimicking the natural carcinogenesis process. Here, we present a protocol for inducing esophageal squamous cell carcinoma (ESCC) in mice using 4NQO. We detail the acquisition of KAT8 esophagus-specific knockout mice, the establishment of 4NQO-induced ESCC, and the validation of the model via western blotting (WB), hematoxylin and eosin (H&E) staining...

Protocol for KAT8 conditional knockout mice generation and its application in 4NQO-induced esophageal tumor model #protocol #starprotocols #cellpress

Protocol for analyzing immune cell infiltration in 3D pancreatic heterotypic spheroids composed of tumor cells and cancer-associated fibroblasts We present a protocol to generate and analyze 3D cancer models composed of tumor cells, cancer-associated fibroblasts (CAFs), and immune cells that mimic the complexity of the pancreatic ductal adenocarcinoma (PDAC) stroma. The protocol includes steps and conditions for establishing 3D co-cultures, embedding for histological analysis, and flow cytometry analysis for the immunophenotyping and characterization of tumor-infiltrating immune cells. These models are valuable for testing strategies and...

Protocol for analyzing immune cell infiltration in 3D pancreatic heterotypic spheroids composed of tumor cells and cancer-associated fibroblasts #protocol #starprotocols #cellpress

Protocol for modeling innate immune training by repeated alum exposure in mice (adjuvant conditioning) We present a protocol to model the effects of innate immune training and reprogramming through repeated alum exposure in mice, enabling the evaluation of immunoregulatory cell populations in both steady-state and inflammatory conditions. We describe steps for integrating adoptive cell transfer, in vivo antigen-specific immunization, and transplant models. We also detail procedures for in vitro stimulation of human peripheral blood mononuclear cells (PBMCs) to explore translational relevance. For...

Protocol for modeling innate immune training by repeated alum exposure in mice (adjuvant conditioning) #protocol #starprotocols #cellpress

Protocol for blastomere injection to generate bilateral hemimosaic Xenopus tadpoles Here, we present a protocol to generate bilateral hemimosaic Xenopus tadpoles that allows systematic, independent manipulation of pre- and postsynaptic neurons in the tadpole retinotectal projection. We describe the steps to perform in vitro fertilization, followed by blastomere injections for two different manipulations. Specifically, we detail procedures for exogenous gene expression (GCaMP6s) by mRNA microinjection and endogenous gene knockdown (GluN1) with antisense morpholino microinjection...

Protocol for blastomere injection to generate bilateral hemimosaic Xenopus tadpoles #protocol #starprotocols #cellpress

Protocol for combining BMP, MEK, WNT inhibition and iNGN2 to enable rapid neuronal differentiation and regional patterning from hiPSCs Neurogenin 2 (iNGN2) overexpression induces human induced pluripotent stem cells (hiPSCs) into neurons but results in a mixed population of peripheral and central nervous system neurons. Pre-differentiation using BMP, MEK, and WNT inhibition (“BMWi”) prior to iNGN2 promotes telencephalic neuron differentiation. We outline neural induction, neural progenitor replating/freezing/thawing, and telencephalic neuron maturation and describe protocols for patterning motor neurons, ventral midbrain, and s...

Protocol for combining BMP, MEK, WNT inhibition and iNGN2 to enable rapid neuronal differentiation and regional patterning from hiPSCs #protocol #starprotocols #cellpress

Protocol for evaluating the economic viability of adiabatic compressed air energy storage systems Techno-economic evaluation is vital for assessing long-duration energy storage. Here, we present a computational workflow evaluating adiabatic compressed air energy storage economic performance. We describe steps for deriving learning-driven cost projections via experience-curve modeling and quantifying uncertainties using Monte Carlo simulations. We then detail procedures for discounted cash flow analysis to determine costs. This protocol enables systematic assessment of storage economic viabil...

Protocol for evaluating the economic viability of adiabatic compressed air energy storage systems #protocol #starprotocols #cellpress

Protocol for generating human tonsil immune organoids with epithelial and germinal center layers using low-Matrigel culture Tonsil immune organoids traditionally include either epithelial layers or immune compartments, failing to recapitulate the integrated physiological microenvironment of human tonsils. Here, we present a protocol to generate tonsil immune organoids that recapitulate both epithelial and germinal center-like structures using a low-concentration Matrigel transwell system. We describe organoid assembly, immune stimulation, and evaluation of epithelial architecture and germinal center formation. This p...

Protocol for generating human tonsil immune organoids with epithelial and germinal center layers using low-Matrigel culture #protocol #starprotocols #cellpress

Protocol for generating high-quality, 45-color spectral flow cytometry data for unsupervised clustering to investigate aging in human PBMCs Spectral flow cytometry enables the use of high-parameter panels. Using a Sony ID7000-optimized 45-color panel, we present a protocol for acquiring and preprocessing high-quality, high-parameter spectral flow cytometry data. We describe steps for batch-learning-assisted comparative cluster identification to study age-induced differences in human peripheral mononuclear cells. We detail spectral flow cytometry procedures, including sample staining, spectral reference acquisition, and unmixing. We ...

Protocol for generating high-quality, 45-color spectral flow cytometry data for unsupervised clustering to investigate aging in human PBMCs #protocol #starprotocols #cellpress

Protocol for isolating plant-derived extracellular vesicles Plant-derived extracellular vesicles (PEVs) are nanosized, lipid bilayer-enclosed vesicles secreted by plant cells into the extracellular space, playing critical roles in intercellular communication. Here, we present a comprehensive and reproducible protocol for isolating PEVs from diverse plant species. Our integrated approach combines differential centrifugation, ultrafiltration, ultracentrifugation, and sucrose density gradient centrifugation to achieve efficient enrichment and purification o...

Protocol for isolating plant-derived extracellular vesicles #protocol #starprotocols #cellpress

Protocol for assessing visual stimulus discrimination in Danionella cerebrum and Danio rerio using habituation/dishabituation Here, we present a protocol to assess visual discrimination in fish, specifically Danionella cerebrum and Danio rerio. We describe steps for the familiarization of subjects to reduce contextual novelty and habituation through repeated presentation of a constant feature stimulus. We then detail procedures for a dishabituation session that introduces a stimulus change. In this protocol, the recovery of behavioral response provides a measure of change detection and feature discrimination. For compl...

Protocol for assessing visual stimulus discrimination in Danionella cerebrum and Danio rerio using habituation/dishabituation #protocol #starprotocols #cellpress

Protocol to establish glioma stem cell spheroids and an enriched cancer-associated fibroblast population from a single patient tumor specimen Glioblastoma is an aggressive, therapy-resistant brain tumor in which the role of cancer-associated fibroblasts (CAFs) remains poorly defined. We present a protocol to simultaneously establish glioblastoma stem cell (GSCs) spheroids and CAFs from a single patient specimen. We describe steps to dissociate tumor tissue, generate a single-cell suspension, and establish GSCs spheroids and CAFs cultures. This protocol details CAFs enrichment, passaging, phenotypic characterization, and cryopreservati...

Protocol to establish glioma stem cell spheroids and an enriched cancer-associated fibroblast population from a single patient tumor specimen #protocol #starprotocols #cellpress

Protocol for spatial sampling and monitoring infection disease dynamics and pathogen spread in social ambrosia beetle colonies Here, we present a protocol for investigating spatial sampling, infection, and pathogen spread in ambrosia beetle colonies to explore infectious disease dynamics in social organisms. We describe steps for rearing Xyleborus affinis colonies, preparing and performing fungal (Metarhizium) infection assays, and examining vertical and horizontal disease transmission. We then detail procedures for spatial sampling within beetle colonies for adult beetle and progeny distribution, microbial commensal an...

Protocol for spatial sampling and monitoring infection disease dynamics and pathogen spread in social ambrosia beetle colonies #protocol #starprotocols #cellpress

Protocol for Nanodisc single-molecule pull-down assay to detect protein-lipid interactions Phospholipids function as signaling molecules regulating numerous processes within the cellular environment. Identifying and characterizing the protein players involved in lipid-mediated functions remains a central focus. Here, we present a protocol for evaluating lipid-protein interactions using mammalian whole-cell lysates expressing proteins of interest in a single-molecule pull-down (SiMPull) assay. We describe steps for preparing materials and slide chambers, performing SiMPull, and analyzi...

Protocol for Nanodisc single-molecule pull-down assay to detect protein-lipid interactions #protocol #starprotocols #cellpress

Protocol for quantitative analysis of adult zebrafish swimming behavior using DeepLabCut Here, we present a protocol for quantifying adult zebrafish swimming behavior using open-source software. We describe steps for recording and preprocessing swimming behavior videos, labeling and training a DeepLabCut network, and applying the trained model to analyze behavior. We then detail procedures for processing and visualizing behavioral data. This protocol is optimized for single-fish tracking under standardized recording conditions and can be completed using beginner-level computational ...

Protocol for quantitative analysis of adult zebrafish swimming behavior using DeepLabCut #protocol #starprotocols #cellpress

Protocol for isolation of total RNA from mouse whole cochlea using organic solvents Determining gene expression changes in cochlear components is important for understanding the mechanisms underlying hearing loss. Here, we present a protocol for the isolation of total RNA from mouse cochleae across a wide range of ages (2–25 weeks old) using organic solvent extraction. We describe the steps for obtaining the entire cochlea from mice and isolating high-quality total RNA suitable for further reverse transcription and PCR. For complete details on the use and execution of this prot...

Protocol for isolation of total RNA from mouse whole cochlea using organic solvents #protocol #starprotocols #cellpress

Protocol for live-cell imaging of mitochondrial dynamics in adult Drosophila oenocytes Mitochondrial dynamics are essential for cellular homeostasis and can be visualized in adult Drosophila oenocytes using live-cell confocal imaging. Here, we present a protocol for live-cell imaging of mitochondrial dynamics in adult Drosophila oenocytes. We describe steps for fly preparation, dissection of abdominal cuticle to expose oenocytes, and mounting. We then detail procedures for time-lapse acquisition of mitochondria labeled with mitoGFP. Optimized imaging parameters enable reproducible...

Protocol for live-cell imaging of mitochondrial dynamics in adult Drosophila oenocytes #protocol #starprotocols #cellpress

Protocol for establishing a mouse model of bilateral maxillary first molar extraction to study alveolar bone healing Alveolar bone healing following tooth extraction is a complex biological process. Here, we present a protocol for establishing a reproducible mouse model of bilateral maxillary first molar extraction to study alveolar bone healing. We describe steps for surgery, postoperative care, and tissue collection for downstream genomic, histological, and micro-computed tomography (CT) analyses. We provide practical strategies to minimize root fracture and soft tissue injury, ensuring procedural consistenc...

Protocol for establishing a mouse model of bilateral maxillary first molar extraction to study alveolar bone healing #protocol #starprotocols #cellpress

Spatiotemporal analysis of cell division during symbiotic root nodule development in the model legume Medicago truncatula This protocol combines rhizobial spot inoculation with deep-tissue imaging to capture cellular processes during early nodule development in the legume Medicago truncatula. We describe steps to visualize DNA replication activity and cell geometry as indicators of cell proliferation and cell expansion processes. We detail steps for rhizobial spot inoculation of seedlings, incubation in 5-ethynyl-2′-deoxyuridine (EdU) medium, sample fixation and labeling of replicated DNA, clearing, staining of the...

Spatiotemporal analysis of cell division during symbiotic root nodule development in the model legume Medicago truncatula #protocol #starprotocols #cellpress

Protocol to perform cell-type-specific transcriptome-wide association study using scPrediXcan framework The scPrediXcan framework enables cell-type-specific transcriptome-wide association studies (TWASs) by integrating deep learning-based prediction of gene expression from DNA sequence and epigenetic features. We present a protocol for scPrediXcan: training cell-type-specific models for expression prediction, predicting personalized expression, and testing associations with genome-wide association study (GWAS) summary statistics. This framework produces scalable TWAS models for different cellular ...

Protocol to perform cell-type-specific transcriptome-wide association study using scPrediXcan framework #protocol #starprotocols #cellpress