#CellPress

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18 hours ago

Protocol for recording visual maps in the mouse superior colliculus and visual cortex with intrinsic optical imaging Intrinsic optical imaging (IOI) is a powerful approach to record visual-functional maps across the mouse superior colliculus (SC) and primary visual cortex (V1). Here, we present a protocol to map retinotopy and orientation preference in the SC within 15 min using periodic stimulation and Fourier analysis. We also describe how to obtain ocular dominance maps in V1 from independent monocular stimulation. We provide detailed instructions for surgical preparation, imaging setup, and preprocessing t...

Protocol for recording visual maps in the mouse superior colliculus and visual cortex with intrinsic optical imaging #protocol #starprotocols #cellpress

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22 hours ago

Protocol for quality control screening of brain organoid morphology Neural organoids can exhibit variability in both tissue shape and tissue identity. Here, we present a pipeline for rapid, protocol-agnostic quality control screening of brain organoids based on their overall gross morphology. We describe a semi-automated image analysis of organoid size, shape, and texture from 2D bright-field imaging. We provide a reference dataset of brain organoids with complex morphology. We show how to integrate input and reference organoids and perform the unbiased sample s...

Protocol for quality control screening of brain organoid morphology #protocol #starprotocols #cellpress

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1 day ago

Protocol for generation of brain metastasis mouse model using stereotactic intracranial tumor-derived cells and modified intracarotid injection Here, we present a protocol for establishing a reproducible mouse model of brain metastasis. We describe the steps for preparing and injecting tumor cells into the internal carotid artery to achieve direct cerebral circulation delivery. We detail procedures for monitoring and validating brain colonization using magnetic resonance imaging (MRI) and histological analysis (H&E staining) and provide guidance on stereotactically generating, harvesting, and re-injecting intracranial tumors via the int...

Protocol for generation of brain metastasis mouse model using stereotactic intracranial tumor-derived cells and modified intracarotid injection #protocol #starprotocols #cellpress

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1 day ago

Protocol to analyze steps of ribosome biogenesis in nuclear and cytosolic fractions of yeast cells Cell fractionation enables the study of compartment-specific RNA processing events in organisms with complex cell walls. Here, we present a protocol for isolating nuclear and cytosolic fractions from Saccharomyces cerevisiae. We describe spheroplast formation using zymolyase, followed by subcellular fractionation via a sucrose density gradient. Finally, we detail the extraction of high-quality RNA from these fractions. This approach overcomes cell wall barriers to allow the analysis of ribosome ...

Protocol to analyze steps of ribosome biogenesis in nuclear and cytosolic fractions of yeast cells #protocol #starprotocols #cellpress

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1 day ago

Protocol for stable cell line production to express muscle-type nicotinic receptor The adult muscle-type nicotinic acetylcholine receptor (AChR) is essential for neuromuscular transmission but is difficult to produce due to the requirement for coordinated subunit assembly. Here, we present a protocol for generating doxycycline-inducible stable cell lines that co-express all four subunits. We describe steps for lentivirus infection, puromycin selection, fluorescence-activated cell sorting, clonal expansion, and protein expression test. This protocol enables AChR production at q...

Protocol for stable cell line production to express muscle-type nicotinic receptor #protocol #starprotocols #cellpress

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2 days ago

Protocol for immunofluorescence detection and quantitative analysis of pH-dependent transcriptional condensates Acidic pH regulates the assembly of transcriptional condensates containing BRD4 and MED1 in a variety of mouse and human cells. Here, we present a protocol to image and quantify BRD4 condensates in bone marrow-derived macrophages. We describe steps for preparing macrophage growth medium at controlled pH levels, performing immunofluorescence experiments, and acquiring images with Airyscan confocal and STED super-resolution microscopy. We detail image processing pipelines to analyze condensate pro...

Protocol for immunofluorescence detection and quantitative analysis of pH-dependent transcriptional condensates #protocol #starprotocols #cellpress

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2 days ago

Protocol for the quantification of digestive exophagy in cell culture Microglia use digestive exophagy to partially degrade, extracellularly, Alzheimer’s amyloid-beta aggregates that are too large to be phagocytosed. Here, we present a protocol to quantify this mechanism in cell culture. We describe steps for extracting primary microglial cells and preparing amyloid-beta model aggregates. We then detail procedures for measuring lysosomal exocytosis toward, and extracellular degradation of, these deposits using quantitative fluorescence microscopy. We also provide ...

Protocol for the quantification of digestive exophagy in cell culture #protocol #starprotocols #cellpress

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2 days ago

Protocol for large-scale, high-yield, high-purity extracellular vesicle purification from human plasma Extracellular vesicles (EVs) hold promise as biomarkers and drug delivery vehicles; however, their broader use is limited by the lack of effective and scalable purification methods. We present a protocol for purifying EVs from human plasma without ultracentrifugation. The workflow incorporates tangential flow filtration (TFF) for volume reduction, polyethylene glycol (PEG)-based precipitation for enrichment, nuclease treatment to remove extravesicular nucleic acids, and multimodal and affinity c...

Protocol for large-scale, high-yield, high-purity extracellular vesicle purification from human plasma #protocol #starprotocols #cellpress

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3 days ago

Protocol to measure protein half-life in cell culture using heavy water Protein turnover, comprising the continued synthesis and clearance of proteins, is required for protein homeostasis and cell survival. Here, we present a protocol to measure protein turnover half-life in cultured cells using D2O labeling. We describe steps to determine the extent of deuterium exchange in amino acids, perform dynamic labeling, and collect protein samples, followed by mass spectrometry and kinetic analysis. The protocol is suitable for measuring protein half-life under steady-stat...

Protocol to measure protein half-life in cell culture using heavy water #protocol #starprotocols #cellpress

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3 days ago

Protocol for precision cutting and short-term culture of lymph node tissue slices for modeling of innate immune responses ex vivo Secondary lymphoid organs are highly organized for the initiation of immune responses. Here, we present a protocol that enables this microanatomical structure to be preserved during short-term culture, allowing ex vivo modeling of human innate immune responses. We describe the steps for precision cutting of human lymph nodes into live tissue cross-sections that can be cultured for up to 20 h with immune stimuli and analyzed by multiple readouts. For complete details on the use and execution of t...

Protocol for precision cutting and short-term culture of lymph node tissue slices for modeling of innate immune responses ex vivo #protocol #starprotocols #cellpress

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3 days ago

Protocol to annotate dendritic cell maturation types in vivo making use of lipid nanoparticle-based approaches The current panels used to annotate mature dendritic cells (DCs) by flow cytometry do not suffice to deconvolute homeostatic from immunogenic mature DCs. Here, we present a protocol to classify DCs along a homeostatic/immunogenic spectrum making use of flow cytometry and/or transcriptional profiling. We describe steps to steer DC maturation in vivo using a lipid nanoparticle (LNP)-based approach. We detail procedures to formulate, characterize, and generate these LNPs and outline their applicati...

Protocol to annotate dendritic cell maturation types in vivo making use of lipid nanoparticle-based approaches #protocol #starprotocols #cellpress

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4 days ago

A spatially localised cryoinjury protocol to study liver regeneration in zebrafish Here, we present a protocol for studying liver regeneration in adult zebrafish using a hepatic cryoinjury technique. We detail the surgical procedure to induce localized necrosis using a cryoprobe. We then detail steps for liver harvesting and CUBIC-based tissue clearing for three-dimensional imaging. The protocol enables comparison of healthy and injured liver parenchyma within individual animals, recapitulating key features of human chronic liver disease, including fibrosis formation and resol...

A spatially localised cryoinjury protocol to study liver regeneration in zebrafish #protocol #starprotocols #cellpress

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4 days ago

Protocol for inducing cystitis via direct instillation of acrolein into the bladder of mice Mouse models of bladder inflammation, termed cystitis, can be used to study urinary dysfunction and visceral pain in both sexes. Here, we present a protocol for a mouse model of cystitis that is induced by instilling acrolein directly into the urinary bladder using a transurethral catheter. We provide instructions for preparing the acrolein solution and catheterizing female and male mice. We then detail procedures for confirming the voiding dysfunction phenotype using the void spot assay.

Protocol for inducing cystitis via direct instillation of acrolein into the bladder of mice #protocol #starprotocols #cellpress

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4 days ago

Protocol for identifying surface membrane proteins and their associated proteome from mouse cortical neuron cultures by in situ biotinylation Modifications to the plasma membrane proteome reflect neuronal physiological states. Here, we present a protocol to profile surface proteins from mouse cortical neuron cultures using in situ biotinylation. The workflow includes neuron culture, surface labeling, enrichment of biotinylated proteins, and on-beads digestion followed by mass spectrometry, enabling quantitative and comparative analysis of membrane-associated proteins across diverse physiological conditions. For complete details on the...

Protocol for identifying surface membrane proteins and their associated proteome from mouse cortical neuron cultures by in situ biotinylation #protocol #starprotocols #cellpress

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5 days ago

Protocol to uncover the protein interactome of small non-coding vault RNAs through RNA antisense purification coupled to mass spectrometry Vault RNAs (vtRNAs) are a family of four small non-coding RNAs (ncRNAs). Here, we present a protocol to identify protein interactors of vtRNA1-1, vtRNA1-2, and vtRNA1-3 in human lung epithelial cells through RNA antisense purification coupled to mass spectrometry (RAP-MS). We describe steps for using biotinylated vtRNA-specific antisense DNA probes to isolate vtRNA-protein complexes that are covalently crosslinked by treatment with ultraviolet (UV) light, followed by mass spectrometry-based anal...

Protocol to uncover the protein interactome of small non-coding vault RNAs through RNA antisense purification coupled to mass spectrometry #protocol #starprotocols #cellpress

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5 days ago

Protocol for iterative indirect immunofluorescence imaging of frozen mouse intestinal tissues Here, we present a protocol for iterative indirect immunofluorescence imaging (4i) of frozen tissues (Cryo-4i). We describe the steps for mouse intestinal sample preparation and antibody elution with standard reagents. Avoiding antibody conjugation or specialized equipment makes this protocol cost-effective and accessible. It requires two days for setup and one day per imaging round; five rounds allow multiplexed detection of 10 markers in a single section. We also detail procedures for using a ...

Protocol for iterative indirect immunofluorescence imaging of frozen mouse intestinal tissues #protocol #starprotocols #cellpress

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5 days ago

Atopic march protocol involving sequential skin and lung inflammation elicited by epicutaneous Staphylococcus aureus and allergen exposure The prevalence of allergic diseases is rising globally, and these diseases often follow a sequential pattern of disease onset in childhood, beginning with atopic dermatitis, followed by food allergy, asthma, and allergic rhinitis. Here, we present a protocol for modeling this “atopic march” in mice. We describe the steps for inducing atopic dermatitis-like skin inflammation through Staphylococcus aureus epicutaneous exposure. We then detail procedures for eliciting lung inflammation via skin all...

Atopic march protocol involving sequential skin and lung inflammation elicited by epicutaneous Staphylococcus aureus and allergen exposure #protocol #starprotocols #cellpress

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6 days ago

Protocol for generating a 3D hydrogel-based tumor resection model in vitro using pancreatic cancer cells We present a protocol for the generation of two distinct in vitro tumor resection models to evaluate cellular responses at tumor margins in 3D. We describe steps for patterning pancreatic cancer cells (Panc-01) embedded in hydrogels using a custom 96-pin metal lid to create standardized resection cavities. In a second model, we detail procedures for refining the protocol to imitate narrow surgical resection margins. This protocol supports diverse treatment modalities and enables reproducible, hi...

Protocol for generating a 3D hydrogel-based tumor resection model in vitro using pancreatic cancer cells #protocol #starprotocols #cellpress

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6 days ago

Protocol to build open-source Discrimin8 maze to study discrimination of reward-context associations in mice during open-field foraging The hippocampus is known to process context-specific memories. We provide a protocol for studying context discrimination in mice utilizing the open-source Discrimin8 maze. We describe the steps for building the maze and using custom-written codes implementing the task. Finally, instructions are provided to train mice on the task. This protocol implements a low-cost, automated maze allowing for investigation of context discrimination in freely moving mice. For complete details on the use and exec...

Protocol to build open-source Discrimin8 maze to study discrimination of reward-context associations in mice during open-field foraging #protocol #starprotocols #cellpress

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1 week ago

Protocol for quantifying the oxidative cleavage of amyloid-β using oxygen-18 isotope labeling and LC-MS/MS Here, we present a protocol for quantifying the oxidative cleavage of amyloid-β using oxygen-18 labeling coupled with high-resolution LC–MS/MS. We describe procedures for amyloid-β peptide preparation, copper(II)/ascorbate-mediated oxidative cleavage, oxygen-18 incorporation, LC–MS/MS acquisition, and isotopologue-based quantitative data analysis. By incorporating oxygen-18 into newly generated C-termini during metal-driven cleavage and applying defined isotope conditions, this protocol enables ...

Protocol for quantifying the oxidative cleavage of amyloid-β using oxygen-18 isotope labeling and LC-MS/MS #protocol #starprotocols #cellpress

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1 week ago

Protocol for the synthesis and characterization of Ru@PCN@PEI nanoparticles with superoxide dismutase catalytic activity Bimetallic organic frameworks (BMOFs) possess versatile structural and functional features that enable applications in catalysis and biomedicine. Here, we present a protocol to synthesize Ru@PCN@PEI nanoparticles by loading Ru nanoparticles onto PCN-222(Mn) and coating the surface with polyethyleneimine (PEI). We describe steps for precursor preparation, particle size regulation, and surface modification. We detail procedures for morphological characterization by transmission electron microscopy...

Protocol for the synthesis and characterization of Ru@PCN@PEI nanoparticles with superoxide dismutase catalytic activity #protocol #starprotocols #cellpress

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1 week ago

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

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1 week ago

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

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1 week ago

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

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1 week ago

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

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1 week ago

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

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1 week ago

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

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1 week ago

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

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1 week ago

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

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1 week ago

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

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