STAR Protocols最新文献

T cell differentiation and selection in the thymus are pivotal for establishing antigen specificity and shaping the functional repertoire of peripheral T cells. Here, we present an in vitro protocol to investigate the roles of specific target genes during early thymic development in mice utilizing retroviral vectors. We describe steps for retroviral packaging, the isolation of primary murine thymic cells, retroviral transduction, and the subsequent in vitro differentiation of thymocytes. For complete details on the use and execution of this protocol, please refer to Fan Zhao et al.¹.

Organ-on-chip (OoC) technology offers a more relevant gut model in comparison with static in vitro gut models from stable cell lines by replicating the 3D structure, mucus production, fluid dynamics, and mechanical forces, closely simulating in vivo conditions. Here, we present a protocol for developing a mucus-producing gut-on-a-chip model from Caco-2 and HT29-MTX-E12 cells. We describe steps for assessing model viability, evaluating barrier integrity, and employing different microscopy techniques, including confocal microscopy.

Here, we present a protocol to assess the lipogenic phenotype of induced neural stem cells (iNSCs) using stable isotopic tracing. We describe steps for the culture and preparation of iNSCs, labeling with [¹³C₆]-glucose and [¹³C₅, ¹⁵N₂]-glutamine, and the subsequent extraction of metabolites, lipids, and proteins from the same sample. This protocol supports single-specimen, mass spectrometry-based multi-omics workflows and is applicable to steady-state analyses, stable isotope tracing, and characterization of protein post-translational modifications. For complete details on the use and execution of this protocol, please refer to Ionescu et al.¹.

Here, we present a protocol comprising two differentiation techniques to derive dorsal sensory spinal interneurons (dIs) from mouse embryonic stem cells (mESCs) through a neuromesodermal progenitor (NMP) precursor. First, we describe the steps for using retinoic acid (RA) to induce dI4s, dI5s, and dI6s, which mediate pain, itch, touch, and sensorimotor integration. Second, we detail the procedures for generating dI1s, dI2s, and dI3s, which mediate proprioception and mechanosensation, using RA together with bone morphogenetic protein (BMP) 4. For complete details on the use and execution of this protocol, please refer to Gupta et al.¹.

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.