Skeletal muscle is the most abundant tissue in the human body and has a tremendous capability to regenerate in response to muscle injuries and diseases. Induction of acute muscle injury is a common method to study muscle regeneration in vivo. Cardiotoxin (CTX) belongs to the family of snake venom toxins and is one of the most common reagents to induce muscle injury. Intramuscular injection of CTX causes overwhelming muscle contraction and lysis of myofibers. The induced acute muscle injury triggers muscle regeneration, allowing in-depth studies on muscle regeneration. This protocol describes a detailed procedure of intramuscular injection of CTX to induce acute muscle injury that could be also applied in other mammalian models.

Macrophages are a heterogeneous class of innate immune cells that offer a primary line of defense to the body by phagocytizing pathogens, digesting them, and presenting the antigens to T and B cells to initiate adaptive immunity. Through specialized pro-inflammatory or anti-inflammatory activities, macrophages also directly contribute to the clearance of infections and the repair of tissue injury. Macrophages are distributed throughout the body and largely carry out tissue-specific functions. In skeletal muscle, macrophages regulate tissue repair and regeneration; however, the characteristics of these macrophages are not yet fully understood, and their involvement in skeletal muscle aging remains to be elucidated. To investigate these functions, it is critical to efficiently isolate macrophages from skeletal muscle with sufficient purity and yield for various downstream analyses. However, methods to prepare enriched skeletal muscle macrophages are scarce. Here, we describe in detail an optimized method to isolate skeletal muscle macrophages from mice. This method has allowed the isolation of CD45⁺/CD11b⁺ macrophage-enriched cells from young and old mice, which can be further used for flow cytometric analysis, fluorescence-activated cell sorting (FACS), and single-cell RNA sequencing.

Inducing loss of function of a target protein using methods such as gene knockout is a powerful and useful strategy for analyzing protein function in cells. In recent years, the CRISPR/Cas-9-based gene knockout technology has been widely used across a variety of eukaryotes; however, this type of simple gene knockout strategy is not applicable to essential genes, which require a conditional knockout system. The auxin-inducible degron (AID) system enables rapid depletion of the target protein in an auxin-dependent manner and has been used to generate conditional mutants in various eukaryotic cell lines. One problem with the AID system is the use of high auxin concentrations for protein degradation, which can cause cytotoxicity. Recently, we established a super-sensitive AID (ssAID) system that allowed a reduction in the amount of auxin required by more than 1,000-fold. We also utilized a single-step method to generate AID-based conditional knockout cells with a ssAID system in various cell lines. In this protocol, we introduce our improved method, which provides a powerful tool for the investigation of the roles of essential genes.

Macrophages are a heterogeneous class of innate immune cells that offer a primary line of defense to the body by phagocytizing pathogens, digesting them, and presenting the antigens to T and B cells to initiate adaptive immunity. Through specialized pro-inflammatory or anti-inflammatory activities, macrophages also directly contribute to the clearance of infections and the repair of tissue injury. Macrophages are distributed throughout the body and largely carry out tissue-specific functions. In skeletal muscle, macrophages regulate tissue repair and regeneration; however, the characteristics of these macrophages are not yet fully understood, and their involvement in skeletal muscle aging remains to be elucidated. To investigate these functions, it is critical to be able to efficiently isolate macrophages from skeletal muscle with sufficient purity and yield for various downstream analyses. Here, we describe in detail an optimized method to isolate skeletal muscle macrophages from mice. This method has allowed the isolation of high-purity CD45⁺/CD11b⁺ macrophages from young and old mice, which can be further used for flow cytometry analysis, fluorescence-activated cell sorting (FACS), and single-cell RNA sequencing.

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a routine procedure in the lab; however, epigenome-wide quantitative comparison among independent ChIP-seq experiments remains a challenge. Here, we contribute an experimental protocol combined with a computational workflow allowing quantitative and comparative assessment of epigenome using animal tissues.

Chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq) is a powerful technology to profile genome-wide chromatin modification patterns and is increasingly being used to study the molecular mechanisms of brain diseases such as drug addiction. This protocol discusses the typical procedures involved in ChIP-seq data generation, bioinformatic analysis, and interpretation of results, using a chronic cocaine treatment study as a template. We describe an experimental design that induces significant chromatin modifications in mouse brain, and the use of ChIP-seq to derive novel information about the chromatin regulatory mechanisms involved. We describe the bioinformatic methods used to preprocess the sequencing data, generate global enrichment profiles for specific histone modifications, identify enriched genomic loci, find differential modification sites, and perform functional analyses. These ChIP-seq analyses provide many details into the chromatin changes that are induced in brain by chronic exposure to cocaine, and generates an invaluable source of information to understand the molecular mechanisms underlying drug addiction. Our protocol provides a standardized procedure for data analysis and can serve as a starting point for any other ChIP-seq projects.

Transfer of mature T cells into immunodeficient mice results in sub-optimal reconstitution of the peripheral T cell pool. Under lymphopenic conditions, dendritic cells are released from tonic control by regulatory T cells (Tregs), and consequently drive activation and proliferation of low affinity T cells specific for endogenous antigens. This oligoclonal proliferation results in a T cell population dominated by T cells possessing an effector/memory phenotype and a limited TCR repertoire. Oligoclonal expansion can be prevented by selectively reconstituting the Treg compartment prior to T cell transfer (Bolton et al., 2015). Reconstitution of the Treg compartment of lymphopenic mice has been tested in immunodeficient mouse strains such as Rag-1^-/-or Rag-2^-/- mice, and in immunosufficient mice rendered transiently lymphopaenic by lethal whole body irradiation as conditioning for bone marrow transplantation (BMT). Transfer of purified Tregs into these hosts, combined with treatment with exogenous IL-2 for 7 days, is sufficient to reconstitute the Treg compartment and reduce expression of dendritic cell costimulatory molecules, a critical process in preventing inappropriate expansion of self-reactive T cells. T cells transferred after Treg reconstitution do not undergo rapid spontaneous proliferation, and instead undergo slow homeostatic division to repopulate the T cell pool with naive T cells, thus allowing optimal reconstitution of peripheral T cell pool.

The detailed protocol is used to isolate different cell types from murine brain as glial cells, including microglia, using an enzymatic digestion that minimizes cellular mortality. A Percoll gradient (30% to 80%) separation allows a maximal recovery of isolated murine microglial cells prior to flow cytometry analysis.

Fluoro-Jade is a fluorescent derivative used for histological staining of degenerating neurons. This technique is simple and sensitive enough to label distal dendrites, axons, axon terminals as well as neuronal bodies. Fluoro-Jade has excitation and emission peak of 480 and 525 nanometer respectively. It can be visualized using a fluorescein/FITC filter. Some reports have demonstrated that Fluoro-Jade can also be useful to detect glial cell death (Anderson et al., 2013; Damjanac et al., 2007).

Macrophages are an essential cell population of innate immunity that plays important roles in inflammatory processes. Two main different phenotypes have been described with opposing activities: The classically activated macrophages (M1) and the alternatively activated macrophages (M2). Alternative activation of mouse macrophages can be induced by type 2 cytokines such as IL-4 and it is characterized by the regulation of the L-arginine metabolism. M2 macrophages convert arginine to ornithine and urea through the action of Arginase-1. Here we described a method for the isolation of peritoneal macrophages from thioglycollate-elicited mice and alternative activation by stimulation with IL-4. Intraperitoneal injection of thioglycollate elicits large numbers of macrophages into peritoneal cavity.