STAR Protocols最新文献

Fibro-adipogenic progenitors (FAPs) are key regulators of skeletal muscle regeneration and influence myogenic differentiation. Here, we present a protocol for the isolation of primary FAPs from injured murine skeletal muscle and the co-culture of C2C12 myoblasts with either 3T3-L1 or primary FAPs. We describe steps for muscle injury, harvest, and digestion followed by FAP isolation. We then detail procedures for co-culture and for assessing myogenic differentiation using immunofluorescence imaging, enabling direct comparison of stromal influences on myoblast differentiation. For complete details on the use and execution of this protocol, please refer to Norris et al.¹.

We present a protocol to isolate viable protoplasted cells of the basidiomycete fungal mutualist (Leucoagaricus gongylophorus) obligately farmed by leafcutter ants. We describe steps for culturing actively growing mycelia, enzymatic digestion of fungal cell walls under osmotic stabilization, and purification of protoplasts (plasma membrane enclosing cytoplasm and organelles). We then detail procedures for assessing viability and wall removal using microscopy and fluorescent staining.

Conditional mutagenesis using site-specific recombinases is widely used in transgenic mouse models; however, its efficiency in vitro is limited by poor tamoxifen activity. Here, we present an optimized in vitro system for Cre-mediated loxP/loxP deletion in primary colonic fibroblasts. First, we describe steps for isolating, culturing, and monitoring murine colonic fibroblasts. Second, we detail procedures for inducing recombination using transducible TAT-Cre recombinase, enabling controlled gene activation or deletion in vitro. For complete details on the use and execution of this protocol, please refer to Chulkina et al.¹.

RNA interference (RNAi) is a promising new approach for oncogene targeting for "undruggable" targets, including KRAS. Here, we present a protocol for evaluating mutant selectivity of KRAS small interfering RNAs (siRNAs) using orthogonal in vitro and in vivo techniques. We describe steps for structural analyses of siRNA complexes, utilization of isogenic HA- and luciferase-tagged cell lines, RNA sequencing for off-target effects, and in vivo evaluation of mutant selectivity. This protocol has potential application for the development of mutant-specific siRNA molecules targeting any oncogene. For complete details on the use and execution of this protocol, please refer to Stanland et al.¹.

Here, we present a protocol to isolate GFP-positive nuclei from whole lung tumor tissue from a genetically engineered mouse model (GEMM) for downstream analysis. First, we outline the procedure for the isolation of fixed GFP-positive nuclei for utilization in chromatin immunoprecipitation followed by sequencing (ChIP-seq) assay. We also describe the sorting of unfixed nuclei for whole-genome methylation analysis. This nuclei collection methodology can be adapted for the retrieval of other fluorescently tagged nuclei and/or for use in other downstream analysis. For complete details on the use and execution of this protocol, please refer to Gillis et al.¹.

The immune system comprises several specialized cells across multiple organs. Here, we present a protocol for the isolation of leukocytes from murine blood, spleen, mesenteric lymph nodes, liver, kidney, and skeletal muscle. We describe steps for cell isolation and gradient density separation and provide four antibody panels to identify adaptive immune cells (e.g., CD4⁺ and CD8⁺ T cell subsets) by flow cytometry. This protocol provides a basic framework for phenotyping immune cells from in vivo mouse models.

Here, we present a protocol for studying how microbiota-derived substrates fuel host mouse metabolism using stable isotope tracing. We describe steps for integrating ¹³C tracing in bacterial culture with ¹³C-labeled CO₂ tracing in free-moving mice. We then detail procedures for determining the metabolic flux and fate of gut microbiota-derived substrates in the host through comparison between germ-free mice and monocolonized or specific pathogen-free mice. This protocol provides a framework for determining metabolic interactions between the gut microbiota and host. For complete details on the use and execution of this protocol, please refer to Fang et al.¹.

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.