Here, we present a protocol to identify nucleotide motifs that predict the pro-inflammatory property of microRNAs (miRNAs) using machine learning. We describe steps for cell culture, miRNA transfection, pro-inflammatory classification, and k-mer discovery. We detail procedures for combining in vitro macrophage assays with exhaustive motif searches and least absolute shrinkage and selection operator (LASSO) regression to define nucleotide sequence features that distinguish pro-inflammatory miRNAs...

Brain-resident T cells act as sentinels, monitoring and supporting immune surveillance and homeostasis. Here, we present a rapid protocol for isolating viable T cells from post-mortem human brain tissue. We describe the process of extracting cells from multiple CNS compartments—including choroid plexus, leptomeninges, dura mater, cerebrospinal fluid, and parenchyma—as well as matched peripheral blood. We detail steps for achieving this through mechanical and enzymatic tissue dissociation, follow...

Type 2 innate lymphoid cells (ILC2s) are a subset of innate lymphoid cells involved in allergic diseases, tissue repair, and tumor immunity. Here, we present a protocol to study the function of immune checkpoint receptors on ILC2s in vitro using plate-bound recombinant ligands. We describe steps for the enrichment, expansion, and purification of human ILC2s from healthy donors’ peripheral blood. For complete details on the use and execution of this protocol, please refer to Ciancaglini et al.1

Advancements in tissue clearing and light sheet microscopy have provided new ways to study neural circuits dedicated to sensory processing in a diverse range of animal species. Here, we present a modified iDISCO+ protocol for whole-brain immunolabeling with the neural activity marker C-FOS in the pigeon brain. We describe steps for whole-brain pre-processing, bleaching, and immunostaining. We then detail procedures for obtaining ultramicroscopic light-sheet images in cleared pigeon brains. For c...

Here, we present a protocol for implementing scalable, modular hardware and software infrastructure to remotely operate programmable miniaturized networks of wireless neural devices in mice. We describe steps for fabricating remote control module (RCM) hardware, software setup, stereotaxic surgery, and probe implantation. We then detail procedures for locomotor, food intake, and social interaction assays. These techniques help enhance automation and throughput for studies of the neurophysiologic...

The phosphate-locking loop (PLL), stabilizing Cas9-DNA interactions, is a key target for optimizing efficiency and specificity. Here, we present a protocol for enhancing Cas9 efficiency and fidelity through structure-guided PLL engineering. We describe steps for identifying PLL engineering targets through sequence alignment and structural analysis, constructing variants via inverse PCR, evaluating efficiency using amplicon sequencing, and assessing specificity through Genome-wide Unbiased Identi...

Here, we present a protocol for inducing oxidative stress in the Drosophila melanogaster larval brain to facilitate the screening of regulators involved in redox homeostasis. We describe steps for the collection of synchronized larvae and their treatment with oxidative stressors. We then detail procedures for larval brain dissection, lipid droplet staining, image acquisition, and the assessment of glutathione redox status. For complete details on the use and execution of this protocol, please re...

Neuronal ensembles are groups of neurons with coordinated activity that are related to a specific brain function. Here, we present a protocol to identify and compare neuronal ensembles from neuronal activity data obtained through two-photon microscopy or electrophysiological recordings using a unified graphical user interface (Ensembles Comparison and Recognition [ENCORE]). We describe steps for installing ENCORE, data loading, neuronal ensemble identification, inspecting results, and comparison...

Fatty acid metabolites, such as eicosanoids and docosanoids, play important biological roles and are strictly regulated by diverse enzymes. Here, we present two approaches for purifying their metabolites, including the phospholipid mediator platelet-activating factor (PAF), for quantification using liquid chromatography-mass spectrometry (LC-MS). We describe steps for tissue collection, lipid extraction, and lipid mediator purification. We then detail procedures for data analysis. This protocol ...

Quantification of immune cells and their activation status in a spatially specific manner in a healthy or diseased tissue is a valuable tool in immunology. Here, we present a protocol for the spatial profiling of immune cells in the mouse cornea and conjunctiva. We describe steps for immunostaining with photocleavable oligonucleotide-conjugated antibodies, selecting regions of interest (ROIs), and collecting UV-cleaved oligonucleotides using GeoMx DSP. We then detail procedures for hybridizing c...

Targeted genome editing using CRISPR-Cas, ZFNs, or TALENs enables precise gene function studies but often produces point mutations or insertions or deletions (indels) that are difficult to detect by conventional PCR. We developed a parallel qPCR assay with an iGenotype index for simple, reliable genotyping. iGenotype values (1, 0, −1) remained constant across allele-specific primers. qPCR data can be analyzed via an R program, enabling large-scale or automated genotyping. For complete details on...

Surface markers for the identification and potential targeting of senescent cells are limited and typically assessed via flow cytometry. Here, we present a protocol to quantify cell-surface vimentin (CSV) as a membrane-associated marker of senescent chondrocytes using a cell-based ELISA. We describe the steps for primary chondrocyte culture and chemical induction of senescence. This protocol offers a high-throughput screening method to simultaneously quantify CSV across multiple samples with min...

Here, we present a protocol to generate an allo-transplantable tumor in mouse models using the YUMM1.7 cell line. We describe steps for mouse breeding using syngeneic C57BL/6 and allogeneic BALB/c mice, injecting the YUMM1.7 melanoma cell line into mice, and collecting and dissociating the growing tumors into single cells. We then detail the procedures for injecting the cells into mice for the generation of an allo-transplantable tumor by progressive passaging into more mismatched hosts. For com...

Neuronal synapses contain rich repertoires of RNAs that can be used for onsite translation and modulation of their proteomes. Here, we present a protocol to recover a synaptosome-enriched fraction adapted to presynaptic RNA profiling, starting from adult Drosophila heads. We describe steps for generating synaptosomes through gentle lysis, followed by centrifugation and a density-based sucrose gradient. We then detail the procedures for validation of the protocol. This protocol can be applied to ...

Human pancreatic slices provide an organotypic platform that preserves native cell interactions for functional studies under near-physiological conditions. We present a protocol to regenerating human pancreatic slices, including lentiviral and adenoviral transduction for lineage tracing and a dual-reporter HIP-Cre system to label neogenic β-cells. The workflow includes tissue dissociation for single-cell analysis and calcium influx measurements to assess glucose responsiveness, enabling identifi...

This protocol details the surgical procedure for genetically manipulating cervical lymph node (LN)-innervating nociceptive neurons in mice. We describe steps for exposing cervical LNs, microinjecting retrograde adeno-associated viruses (AAVs), and verifying viral transduction in the trigeminal ganglia. This approach enables specific labeling, chemogenetic inhibition, or gene knockout in LN-innervating neurons to study neuroimmune crosstalk. For complete details on the use and execution of this p...

Optogenetic modulation of peripheral sensory nerve activity holds great potential for revealing the mechanisms underlying sensory disorders, with important implications for developing therapeutic strategies. Here, we present a protocol for applying optogenetic techniques to peripheral sensory nerves using an adeno-associated virus (AAV) vector. We describe the procedure for gene transduction into dorsal root ganglion neurons via retrograde transport following intra-nerve AAV injection. We then o...

Here, we present a protocol for describing the differentiation of human cortical neurons from induced pluripotent stem cells (iPSCs) and the subsequent analysis of lipid membrane fluidity using the LipiORDER fluorescent probe. We describe steps for embryoid body formation, neuronal induction with adherent culture maturation, and live-cell membrane fluidity measurement. This approach allows for investigation of the effects of lipid membrane fluidity on neuronal function and pathophysiology. For c...

We present a high-sensitivity fluorescence anisotropy-based protocol (fluorescence anisotropy single-stranded targeting [FASST]) to monitor in vitro ligand-induced conformational switching of riboswitches. We first describe the sequence design of single-stranded fluorescent DNA probes that selectively bind to specific riboswitch conformational states. Next, we describe DNA probe binding affinity and conformational switching measurements using a 96-well plate reader. We then detail the procedures...

Here, we present a protocol combining quantitative micro-elastography (QME) with transmitted light (TL) and immunofluorescence (IF) microscopy to study ovarian elasticity and composition in situ. We describe steps for excising murine ovaries, acquiring QME scans, and fixing the tissue. Subsequently, the tissue is sectioned and imaged with TL microscopy to locate functional components, such as corpora lutea and follicles, and segment them within the QME data. Finally, IF is used to analyze cell a...

Hyaluronic acid is a ubiquitous glycosaminoglycan with numerous biological and structural functions in the body. Here, we present a protocol for the practical and efficient functionalization of HA with tyramine. We describe the reaction conditions for its DMTMM-mediated coupling, the subsequent purification and drying steps, and product characterization through 1H NMR and UV spectrophotometry. This protocol produces materials which can form hydrogels with tunable viscoelastic properties, for a r...

We present a protocol to measure transcriptional bursting of endogenous human genes that is widely applicable to adherent cells. We describe the steps for nascent RNA fluorescence in situ hybridization (nscRNA-FISH) in a high-throughput imaging format. We then detail the procedures for automated microscopy and analysis to measure bursting behavior of genes in adherent cell lines. The frequency of bursting alleles in a population is used as a readout for bursting behavior. For complete details on...

Renal cancer is characterized by an insidious onset and a high tendency for lung metastasis in advanced stages. Here, we present a protocol for constructing an orthotopic mouse model that recapitulates the spontaneous metastasis process of human renal cell carcinoma (RCC). We describe steps for dynamically monitoring tumor growth and metastasis by in vivo bioluminescence imaging. We then detail the procedures for performing histopathological analyses. This protocol provides a reliable preclinica...

Microglia, the resident phagocytes of the central nervous system, clear diverse substrates in development, aging, injury, and disease. Here, we present a protocol to visualize phagocytosed content within microglial lysosomes in the rodent brain using immunohistochemistry and confocal microscopy. We describe the steps for tissue staining, image acquisition, and lysosome analysis. We then detail the procedures for microglia and lysosome reconstruction for 3D visualization. For complete details on ...

Calcium (Ca2+) signaling is essential to cellular processes, such as signal transduction and migration, and dysregulation of Ca2+ homeostasis can impact cellular function. Here, we present a protocol to analyze Ca2+ signaling by its release into the cytosol in response to and after metabolic stimulation. We describe the steps for cell starvation from excessive nutrients and stimulation, followed by flow cytometry-based and microscopic analysis of calcium signaling. This protocol enables analysis...

Here, we present the methodology for surgical preparation of the mouse molar teeth and subsequent adeno-associated virus (AAV) transduction of intradental neurons within the tooth (AAV-i.d.). We describe steps for mouse positioning, microscope setup, drilling to expose pulp horns, and applying the AAV-silk mixture. We then detail procedures for composite placement to cover the preparation and trigeminal ganglion (TG) screening to visualize the surface and assess and verify AAV labeling. AAV-i.d....

Up to half of human disease-causing variants may disrupt RNA splicing, but their computationally predicted effects require experimental validation. Here, we present a protocol to test splice-altering variants using full-length gene reporters. We describe steps for reporter construct design, site-directed mutagenesis, delivery into cells, and isoform analysis. Full-length reporters offer key advantages over minigenes, particularly for clinically relevant genes with compact loci, by preserving nat...

The functional profiling of intestinal epithelial cells (IECs), which comprise multiple heterogeneous populations, is crucial for understanding host interactions with dietary and microbial factors. Here, we present a protocol for dissociating mouse epithelium based on TrypLE Express and Dispase II. We describe steps for tissue collection, crypt isolation, and enzyme digestion. This protocol enables the recovery of millions of viable IECs from the mouse intestine and is particularly useful for is...

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...

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...

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...

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 ...

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...

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...

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 ...

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...

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...

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...

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...

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...

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.

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...

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...

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 ...

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...

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...

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...

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 ...

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...