STAR Protocols最新文献

Here, we present a protocol for quantitatively assessing the microstructure of nuclear fiber layers by immunolabeling of a nuclear skeleton protein lamin A through structured illumination microscopy (SIM) coupled with an innovative algorithm in Arivis 4D. We describe steps of sample preprocessing for SIM and imaging of the inner-nucleus skeleton network of lamin A. We then detail procedures for creating a novel algorithm in Arivis 4D software to quantitatively assess the microparameters of the nuclear skeleton in a high-resolution image.

Adeno-associated viruses (AAVs) are versatile, non-integrating vectors for in vivo gene delivery. We present a reproducible workflow for generating Cre-dependent FLEX-AAVs, quantifying viral titer, and performing localized injections for cell-type-specific transgene expression in mice. The protocol also details the assessment of thermogenic capacity in genetically modified brown adipocytes using Clark-type electrode respirometry. For complete details on the use and execution of this protocol, please refer to Bunk et al.¹.

The device for axon and cancer cell interaction testing (DACIT) mimics neuron-cancer interactions by compartmentalizing neuron soma and axons. Here, we present a protocol to design, fabricate, load, and image the DACIT. We describe steps for DACIT master generation, smooth-on mold replication, and DACIT fabrication. We then detail procedures for cell loading in 2D and 3D settings. This protocol also includes custom 3D-printed imaging holders. For complete details on the use and execution of this protocol, please refer to Velazquez-Quesada et al.¹.

Growth of cells in 3D cultures can more accurately predict in vivo behavior than the traditional 2D culture system, which lacks the complex environment of natural tissue. In this protocol, we provide steps to generate 3D collagen cultures in a 384-well format suited for high-throughput drug screens. We also detail our use of this protocol to assess morphological changes to 3D colonies of SC colorectal cancer cells, which serve as a robust readout for drug response. For complete details on the use and execution of this protocol, please refer to Harmych et al.¹.

The dynamics of the early steps of protein aggregation remain poorly understood, particularly in the case of α-synuclein (α-syn) aggregation, the hallmark of synucleinopathies. Here, we present a protocol that combines light-inducible protein aggregation (LIPA) with proximity biotinylation using an UltraID construct. We describe the workflow from protein expression to biochemical validation, including the purification of biotinylated proteins prior to liquid chromatography-mass spectrometry (LC-MS) analysis and subsequent validation. This platform provides a powerful strategy to identify proteins interacting with nascent α-syn aggregates. For complete details on the use and execution of this protocol, please refer to Teixeira et al.¹.

Complete spinal cord injury (SCI) leads to irreversible neurological damage due to failed neural repair, with no effective therapies currently available. Here, we present a protocol to induce severe contusive-compressive SCI at thoracic T11 level in mouse using the NYU-MASCIS II impactor. We describe steps for performing laminectomy, inducing the injury, and validating it through functional and histological analysis. This protocol replicates key aspects of human secondary injury, making it valuable for preclinical testing of SCI therapies.

Understanding changes in gene expression and cell-cell signaling among spatial regions in diseased tissues adds critical biological information to understanding mechanisms. Here, we present a protocol to investigate molecular transcriptional drivers within intact murine tissue using 10× Genomics Visium spatial transcriptomics and 10× Genomics FLEX single-cell RNA sequencing (scRNA-seq) data. We describe steps for collecting mouse brain tissue from multiple ages, processing samples, and mounting the tissue. We then detail procedures for staining, FLEX tissue section collection, fixation, dissociation, and cell storage. For complete details on the use and execution of this protocol, please refer to Burns et al.¹.

Here, we present a protocol for evaluating glucose metabolism in mouse retinas and retinal pigment epithelium (RPE)-choroid tissue by tracking the incorporation of ¹³C₆ from U-¹³C₆-glucose with gas chromatography-mass spectrometry (GC-MS). We describe steps for incubating tissues in Krebs-Ringer bicarbonate solution and homogenizing tissues. We then detail procedures for extracting metabolites and determining isotopic labeling of intermediates in glycolysis and the tricarboxylic acid (TCA) cycle using GC-MS. The approach has been adapted to study glucose metabolism in various tissues, animal models, and genetic conditions. For complete details on the use and execution of this protocol, please refer to Nolan et al.¹.

RNA-protein interactions drive gene regulation, subcellular organization, and noncoding RNA function. Here, we present a protocol for measuring RNA-protein associations in formaldehyde-crosslinked mammalian cells using RNA immunoprecipitation followed by sequencing (RIP-seq) and quantitative PCR (RIP-qPCR). We include steps and best practices for qualifying reagents, preparing cells, and processing and analyzing data, including a standardized set of steps to quantify signal over noise. This protocol is broadly applicable for the study of RNA-protein interactions in cells. For complete details on the use and execution of this protocol, please refer to Trotman et al.¹.

Spatial analysis of cells and their microenvironment within tissues enhances our understanding of biological processes. Ideally, a broad range of biomolecules should be analyzed in large 3D tissue specimens at subcellular resolution. Here, we present a protocol to identify and extract target sections from previously cleared tissues. We describe steps for combining 3D light sheet imaging and subsequent 3D-guided deep cell phenotyping via multi-cyclic 2D microscopy.