STAR Protocols (@cp-starprotocols)

Protocol to generate immunocompetent human heart-macrophage assembloids for the study of immune-cardiac interactions Here, we present a protocol to generate fully human pluripotent stem cell (hPSC)-derived human heart-macrophage assembloids. We describe steps for differentiating hPSCs into monocytes, generating cardiac organoids, integrating CD14+ monocytes to become cardiac tissue-resident macrophages, and maturing the assembloids with metabolic cues. Furthermore, we provide a use-case example of disease modeling for the study of atrial arrhythmias. The system provides a reproducible, scalable, and efficient ...

Protocol to generate immunocompetent human heart-macrophage assembloids for the study of immune-cardiac interactions #protocol #starprotocols #cellpress

06.03.2026 22:09 👍 0 🔁 0 💬 0 📌 0

Protocol to reconstitute motor protein clustering on vesicle membranes using a DNA scaffold-based system in vitro Cytoskeletal motor proteins drive long-range intracellular transport, but how their organization on vesicle membranes affects motility remains poorly understood. Here, we present a protocol to reconstitute kinesin-1 clustering on synthetic liposomes using a DNA-based scaffold. We describe steps for functionalizing liposomes with kinesin-1, inducing DNA-mediated motor clusters, and analyzing liposome motility on surface-immobilized microtubules. This protocol enables quantitative investigation of...

Protocol to reconstitute motor protein clustering on vesicle membranes using a DNA scaffold-based system in vitro #protocol #starprotocols #cellpress

06.03.2026 20:26 👍 0 🔁 0 💬 0 📌 0

Protocol for single-cell optimization objective and trade-off inference Single-cell optimization objective and trade-off inference (SCOOTI) is a computational framework that integrates bulk and single-cell omics data with genome-scale metabolic modeling to infer metabolic objectives and trade-offs in biological systems. Here, we present a protocol for installing and running SCOOTI, using transcriptomics, proteomics, and metabolomics data to constrain metabolic models. We describe steps to interpret metabolic priorities across different cell states or conditions thro...

Protocol for single-cell optimization objective and trade-off inference #protocol #starprotocols #cellpress

06.03.2026 16:08 👍 0 🔁 0 💬 0 📌 0

Protocol for mouse vascular dementia model and evaluation of progressive tissue damage in subcortical white matter and adjacent cortex Here, we present a protocol for generating a vascular dementia (VaD) mouse model through stereotaxic microinjection of a vasoconstrictor into the subcortical white matter (WM). We describe steps for preparing the N5-(1-iminoethyl)-L-ornithine (L-NIO) solution, surgical procedures, and immunohistochemistry. We then detail procedures for confocal imaging and image analysis using ImageJ and Imaris software to evaluate key pathological features of human VaD, including vessel dilation, demyelination,...

Protocol for mouse vascular dementia model and evaluation of progressive tissue damage in subcortical white matter and adjacent cortex #protocol #starprotocols #cellpress

04.03.2026 16:08 👍 0 🔁 0 💬 0 📌 0

Protocol for MR1-ligand cross-linking to discover metabolite antigens by proteomics Here, we present a protocol for the discovery of MR1-bound metabolite ligands by stabilizing Schiff base interactions between MR1 and its ligands. We describe steps for enriching MR1-ligand complexes using MR1-expressing A549 cells and covalently cross-linking ligands to the MR1 lysine 43 residue via reductive amination. We then detail procedures for identifying ligand-modified MR1 peptides by mass spectrometry and for further validation of candidate ligands using MR1 surface upregulation and TC...

Protocol for MR1-ligand cross-linking to discover metabolite antigens by proteomics #protocol #starprotocols #cellpress

03.03.2026 22:08 👍 0 🔁 0 💬 0 📌 0

Protocol for establishing a chemoresistant orthotopic triple-negative breast cancer patient-derived xenograft model for preclinical drug testing Here, we present a protocol for establishing patient-derived xenograft (PDX) mouse models of chemoresistant triple-negative breast cancer (TNBC). We describe steps for tumor collection, surgical implantation, and serial expansion. We then detail procedures for histopathological validation and in vivo therapeutic assessment to confirm the maintenance of chemoresistance across passages. These models provide a clinically relevant platform to investigate mechanisms of treatment resistance, evaluate ...

Protocol for establishing a chemoresistant orthotopic triple-negative breast cancer patient-derived xenograft model for preclinical drug testing #protocol #starprotocols #cellpress

03.03.2026 20:26 👍 0 🔁 0 💬 0 📌 0

Protocol for the precise activation of intradental neurons in mice via electrical stimulation Intradental neurons encode tooth sensation, yet identifying them without sacrificing tooth structural integrity remains challenging. Here, we present a protocol for the non-invasive electrical stimulation of intradental neurons with single-molar tooth resolution in anesthetized mice. We describe details for head fixation to access molars. We then provide steps to deliver direct current (DC) pulses to an individual molar while simultaneously monitoring induced current. The protocol can be combine...

Protocol for the precise activation of intradental neurons in mice via electrical stimulation #protocol #starprotocols #cellpress

03.03.2026 16:08 👍 1 🔁 0 💬 0 📌 1

Protocol to investigate the effects of localized gene expression disruption on mouse limb development via direct siRNA injection and explant culture Here, we present a protocol for localized gene knockdown in embryonic mouse hindlimb explants using direct small interfering RNA (siRNA) injection, combined with a semi-quantitative morphology scoring system to assess developmental outcomes. We describe steps for mating of samples, preparing hindlimb explants for culture and siRNA-Lipofectamine complex, and direct injection of the siRNA-Lipofectamine complex. We then detail procedures for explant culture and maintenance, data analysis, tissue fi...

Protocol to investigate the effects of localized gene expression disruption on mouse limb development via direct siRNA injection and explant culture #protocol #starprotocols #cellpress

02.03.2026 20:26 👍 1 🔁 0 💬 0 📌 0

Protocol for geographical mapping of pesticide contamination Pesticide residues in the environment threaten agricultural and human health. Here, we present a framework for mapping and comparing the contamination levels of pesticides across specific environmental compartments in various regions. We describe steps for applying the pesticide residual concentration and assigning a score to regions by calculating the deviation of pesticide concentrations in each region from the overall central tendency. Comparing scores among regions can help environmental age...

Protocol for geographical mapping of pesticide contamination #protocol #starprotocols #cellpress

02.03.2026 16:08 👍 0 🔁 0 💬 0 📌 0

Protocol for the formulation and characterization of extracellular vesicle-crosslinked supramolecular hydrogels Extracellular vesicles (EVs) can serve as supramolecular crosslinkers to form injectable, intrinsically bioactive hydrogels. This protocol details the formulation, rheological characterization, and analysis of EV-crosslinked hydrogels with tunable mechanics. We describe steps for preparing alkyl-modified hydroxypropyl methylcellulose (HPMC) polymers, mixing polymers with EVs to form supramolecular networks, and assessing properties of the resulting hydrogels. We provide guidance for linking rheo...

Protocol for the formulation and characterization of extracellular vesicle-crosslinked supramolecular hydrogels #protocol #starprotocols #cellpress

01.03.2026 20:27 👍 0 🔁 0 💬 0 📌 0

Protocol for preparing and characterizing samples for combined microsecond freeze-hyperquenching and electron paramagnetic resonance spectroscopy Microsecond freeze-hyperquenching (MHQ) coupled with pulsed electron-electron double resonance spectroscopy (PELDOR) can resolve conformational changes in biomolecules over space and time. Here, we present a protocol for preparing MHQ samples of a cyclic nucleotide-binding domain by rapid mixing with cyclic adenosine monophosphate. Further, we outline the acquisition and analysis of PELDOR data on MHQ samples and study the dynamics of protein-ligand interactions. This protocol can also be extend...

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

01.03.2026 16:08 👍 0 🔁 0 💬 0 📌 0

Computational protocol for hierarchical Bayesian modeling of perception and generalization in fear conditioning Understanding human generalization behavior requires disentangling underlying cognitive and perceptual mechanisms. Here, we present a computational protocol to analyze individual differences in fear generalization by integrating a Bayesian state-space perceptual model with a hierarchical mixture generalization model. We describe steps for applying a state-space model to measure perceptual data and for calculating stimulus distance from these probabilistic representations. We then detail procedur...

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

28.02.2026 20:26 👍 0 🔁 0 💬 0 📌 0

Protocol for efficient CRISPRi-mediated silencing of retrotransposons in human pluripotent stem cells Here, we present a workflow for transcriptional silencing of transposable elements (TEs) in human induced pluripotent stem cells (hiPSCs). We describe steps for designing guide RNAs (gRNAs) to target TE families or unique TE loci. We also detail procedures for validating the efficiency and specificity of large-scale CRISPRi-based silencing using a multiome approach combining bulk RNA sequencing, CUT&RUN epigenetic profiling, and proteomics. This framework optimizes the performance and interpreta...

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

28.02.2026 16:08 👍 0 🔁 0 💬 0 📌 0

Protocol for generating megakaryocytes from patient induced pluripotent stem cells for disease modeling and compound screening Here, we present a protocol for generating megakaryocytes from patient induced pluripotent stem cells (iPSCs). We describe steps for expanding and dissociating iPSCs into single cells and aggregating them into spin embryoid bodies (EBs). We detail procedures for lineage commitment and differentiation toward megakaryocytes within 14 days. This protocol supports phenotypic characterization and compound testing across multiple patient-derived iPSC lines. For complete details on the use and executio...

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

27.02.2026 22:08 👍 0 🔁 0 💬 0 📌 0

Protocol for formulation and evaluation of phytosterol-based lipid nanoparticles as cholesterol alternatives for mRNA delivery We present a protocol for formulating and evaluating lipid nanoparticles (LNPs) that incorporate the plant-derived sterols ginsenoside Rg2 and protopanaxadiol (PPD) as substitutes for cholesterol. The protocol describes mRNA preparation, manual mRNA-LNP encapsulation, and physicochemical characterization. We further detail in vitro and in vivo procedures to evaluate mRNA delivery efficiency. This protocol provides a robust and cost-effective platform for efficient mRNA delivery. For complete det...

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

27.02.2026 20:27 👍 0 🔁 0 💬 0 📌 0

Protocol for the preparation of flexible material from the mycelium of wood-decay fungal strains and mechanical property investigation Extraction of chitin-glucan-based materials from fungi has several advantages over animal-based chitin. Here, we present a protocol for extracting chitin-glucan material from the mycelium of wood-decay fungal strains and investigating its mechanical properties. We describe steps for extracting chitin-glucan-based material, using acetic acid as a crosslinking agent to improve mechanical properties, and adding glycerol to increase flexibility. We then detail procedures for investigating tear stren...

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

27.02.2026 16:09 👍 0 🔁 0 💬 0 📌 0

Protocol for evaluating seedling greening capacity during dark-to-light transition We present a protocol to evaluate greening capacity in etiolated Arabidopsis seedlings during the critical dark-to-light transition. We describe steps for sample preparation and sowing and then detail procedures for quantifying protochlorophyllide accumulation in darkness, the greening rate upon illumination, and reactive oxygen species levels as an indicator of photo-oxidative stress. This protocol can be used for screening and phenotypic quantification across genetic backgrounds. For complete ...

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

26.02.2026 22:08 👍 0 🔁 0 💬 0 📌 0

Protocol for purifying recombinant HBV capsids from HepG2 cells and comparative phosphorylation analysis by phosphate-affinity SDS-PAGE The hepatitis B virus (HBV) capsid protein (HBc or Cp) contains multiple phosphorylation sites that regulate distinct stages of the viral lifecycle. Here, we present a protocol for purifying HBV capsids by gradient ultracentrifugation and analyzing their phosphorylation states. We describe detailed steps for transfection, gradient purification, capsid detection, concentration, and phosphorylation-level profiling. This protocol provides a practical approach to assessing the global phosphorylation...

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

26.02.2026 20:27 👍 0 🔁 0 💬 0 📌 0

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 Tissue-resident memory T cells (TRMs) are a distinct subset of T lymphocytes that permanently localize in non-lymphoid tissues and mediate rapid, localized immune responses. Here, we present a protocol to study human TRMs using an in vitro model system that induces TRM-like T cells. We describe steps for generating the 3D nasal epithelial model and inducing TRMs via co-culture with autologous immune cells. We then detail procedures to analyze TRMs using flow cytometry, single-cell RNA sequencing...

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

26.02.2026 16:08 👍 0 🔁 0 💬 0 📌 0

Protocol for automated analysis of biological images using Python code Image analysis in Python provides greater flexibility and an open-source alternative to commercially available software. In this protocol, we present steps for quantifying features in various common biological images in a manner that is tailored to scientists with little coding background. Using example experiments, we outline steps to read in a variety of image file types and prepare them for quantitative analysis. We then discuss approaches for segmenting images, extracting measurements from s...

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

25.02.2026 16:08 👍 0 🔁 0 💬 0 📌 0

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

24.02.2026 22:08 👍 0 🔁 0 💬 0 📌 0

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

24.02.2026 20:26 👍 0 🔁 0 💬 0 📌 0

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

24.02.2026 16:08 👍 0 🔁 0 💬 0 📌 0

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

23.02.2026 20:27 👍 0 🔁 0 💬 0 📌 0

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

23.02.2026 16:09 👍 0 🔁 0 💬 0 📌 0

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

22.02.2026 20:27 👍 0 🔁 0 💬 0 📌 0

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

22.02.2026 16:09 👍 0 🔁 0 💬 0 📌 0

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

21.02.2026 20:26 👍 1 🔁 0 💬 0 📌 0

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

21.02.2026 16:09 👍 0 🔁 0 💬 0 📌 0

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

20.02.2026 22:08 👍 0 🔁 0 💬 0 📌 0

STAR Protocols

Latest posts by STAR Protocols @cp-starprotocols