STAR Protocols is an open access, peer-reviewed journal from Cell Press. We offer structured, transparent, accessible, and repeatable step-by-step experimental and computational protocols from all are...

STAR Protocols is an open access, peer-reviewed journal from Cell Press. We offer structured, transparent, accessible, and repeatable step-by-step experimental and computational protocols from all are...

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...