Protocol for pro-inflammatory microRNA motif discovery using machine learning

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...

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Protocol for isolating viable human central nervous system T cells

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...

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Protocol to isolate, expand, and activate human type 2 innate lymphoid cells to study immune checkpoint function

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

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Protocol for whole-brain immunolabeling, clearing, and light-sheet imaging of C-FOS in the pigeon

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...

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Protocol to design and implement scalable wireless network system for high-throughput, automated behavioral and circuit neuroscience in mice

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...

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Protocol for enhancing Cas9 efficiency and fidelity through structure-guided phosphate-locking loop engineering

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...

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Protocol for acute oxidative stress induction in third-instar Drosophila melanogaster larvae

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...

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Protocol for identifying and comparing neuronal ensembles using different algorithms within a graphical user interface

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...

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Protocol to purify fatty acid metabolites with two approaches for quantification using liquid chromatography-mass spectrometry

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 ...

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Protocol for spatial profiling of immune cells in the mouse cornea and conjunctiva using NanoString GeoMx DSP and nCounterPro platforms

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...

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Protocol for streamlining genotyping of germline-transmissible mutants from genome editing by using a parallel qPCR-based index and R analysis

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...

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Protocol to quantify cell-surface vimentin on senescent human chondrocytes and other cell types via a cell-based ELISA

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...

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Protocol to generate an allo-transplantable tumor in mouse models using the YUMM1.7 cell line

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...

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Protocol for preparation of a synaptosome-enriched fraction to profile presynaptic RNA content in adult Drosophila brains

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 ...

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Protocol to study regeneration in human pancreatic slices using viral tracing, extended culture, dissociation, and functional Ca2+ influx techniques

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...

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Protocol for genetic manipulation of cervical lymph node-innervating nociceptive neurons in mice via retrograde viral tracing

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...

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Protocol for intra-nerve AAV injection and dorsal root potential recording for optogenetic modulation of the peripheral sensory nerve activity

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...

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Protocol for measuring lipid membrane fluidity in human iPSC-derived neural cells

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...

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Protocol for detecting in vitro riboswitch conformational switching using a fluorescence anisotropy single-stranded RNA-targeting approach

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...

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Protocol to study murine ovarian elasticity and composition in situ by integrating quantitative micro-elastography with light microscopy

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...

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Protocol for preparing tyramine functionalized hyaluronic acid via aqueous DMTMM-mediated conjugation

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...

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Protocol to measure transcriptional bursting of endogenous genes using high-throughput RNA-FISH

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...

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Protocol for constructing an orthotopic mouse model of metastatic renal cell carcinoma

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...

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Protocol for visualizing microglial lysosomal content by immunohistochemistry in the rodent brain

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 ...

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Protocol to assess calcium signaling in response to metabolic alterations by flow cytometry and live-cell imaging

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...

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Protocol for targeted peripheral adeno-associated viral vector transduction of the intradental neurons in mice

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....

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Protocol for validating computationally predicted splice-altering variants using full-length gene reporter assays

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...

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Optimized protocol for dissociation of mouse intestinal epithelium to viable single-cells

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...

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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...

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