Protocol for establishing and evaluating a cancer cachexia mouse model.

Cancer cachexia mouse models are needed to recapitulate the clinical features of patients with cachexia. Here, we present a protocol for the establishment and evaluation of cancer cachexia mouse models. We delineate the steps in preparing tumor cells for inoculation and surgical procedures. After the establishment of these mouse models, we describe essential techniques to assess cancer cachexia, including grip strength evaluation, tissue collection, and the calculation of cross-sectional areas of muscle tissue. For complete details on the use and execution of this protocol, please refer to Liu et al.,1 Yang et al.,2 Shi et al.,3 and Zhou et al.4.

Protocol for analyzing BCG-induced trained immunity in murine bone marrow-derived macrophages.

Bacillus Calmette-Guérin (BCG), the only licensed tuberculosis vaccine, provides non-specific protection against non-tuberculosis diseases that is mediated by trained immunity, a functional reprogramming mediated by innate immune memory. Here, we present a protocol for analyzing BCG-induced trained immunity in murine bone marrow-derived macrophages (BMDMs). We describe steps for preparing BCG single bacterial suspensions, isolating BMDM cells, and the training process. This protocol can assist researchers to conveniently utilize BMDM cells to study trained immunity. For complete details on the use and execution of this protocol, please refer to Xu et al.1.

Protocol for fluorescent live-cell staining of tardigrades.

Tardigrades are microscopic organisms with exceptional resilience to environmental extremes. Most protocols to visualize the internal anatomy of tardigrades rely on fixation, hampering our understanding of dynamic changes to organelles and other subcellular components. Here, we provide protocols for staining live tardigrade adults and other postembryonic stages, facilitating real-time visualization of structures including lipid droplets, mitochondria, lysosomes, and DNA.

Protocol for electrophysiological measurements of circadian changes in excitability in dentate granule cells from adult mice.

Many types of neurons exhibit a daily rhythm of intrinsic excitability. Here, we present a protocol for assessing circadian regulation of dentate granule cell excitability using a mouse model for conditional knockout of the molecular clock protein BMAL1. We describe steps for obtaining healthy oblique horizontal slices that contain the hippocampus and measuring intrinsic excitability and synaptic potentials by combining whole-cell patch-clamp recordings and perforant-path electric stimulation. We then detail procedures for validating single-cell genetic deletion of Bmal1 by immunohistochemistry. For complete details on the use and execution of this protocol, please refer to Gonzalez et al.1.

Protocol for preparing mammalian skin samples encompassing hair follicles for spatial transcriptomics.

Spatial transcriptomics enables a single-cell resolution view of gene expression patterns in tissues, providing insight into their biological functions. However, applying this approach to the skin presents inherent challenges. Here, we present a protocol for preparing mammalian skin samples encompassing hair follicles for spatial transcriptomics. We describe steps for sample preparation, embedding, acquisition of frozen slices, RNA quality control, tissue mounting, fixation, staining, and imaging. We then detail procedures for permeabilization, reverse transcription, and cDNA collection. For complete details on the use and execution of this protocol, please refer to Chen et al.1.

Protocol for transplantation of cells derived from human midbrain organoids into a Parkinson's disease mouse model to restore motor function.

Midbrain organoids provide an innovative cellular source for transplantation therapies of neurodegenerative diseases. Here, we present a protocol for midbrain organoid-derived cell transplantation into a Parkinson's disease mouse model. We describe steps for midbrain organoid generation, single-cell suspension preparation, and cell transplantation. This approach is valuable for studying the efficacy of midbrain organoids as a potential cellular source for restoring motor function. For complete details on the use and execution of this protocol, please refer to Fu et al.1.

Protocol for generation of CRISPR-Cas9-mediated specific genomic insertion of P2A-Gal4 to reveal endogenous gene expression in Drosophila.

Generating a transgene with a reporter inserted into the genome helps us study endogenous gene expression patterns in model organisms. Here, using Drosophila melanogaster, we present a protocol for generating a P2A-Gal4 insertion through CRISPR-Cas9-mediated homology recombination. We describe the design strategy, steps for constructing the injection plasmids, and the fly-cross scheme for screening the transformants from the G0 generation. This protocol can also be applied to introduce mutations or various genetic tools into the fly genome. For complete details on the use and execution of this protocol, please refer to Li et al.1.

Protocol to induce neurodegeneration in a local area of the mouse brain by stereotaxic injection.

In vivo models of brain pathology are crucial for studying neurological diseases. Here, we present a protocol to induce a pathological condition in a mouse brain area by local injection of neurotoxic stimulus. We describe steps for preparing reagents, stereotaxic injection procedures to induce neurodegeneration in the hippocampus, and preparation of brain sections to examine the induced model. This protocol is useful for studying how local pathology affects other brain areas and neighbor cells and its functional consequences in behavior. For complete details on the use and execution of this protocol, please refer to Zhang et al.1.

Protocol for analyzing arginase I expression in tumor-associated myeloid-derived suppressor cells from murine colon cancer using flow cytometry.

Arginase1 (ARG1) is a metabolic enzyme that is highly expressed in tumor-associated myeloid-derived suppressor cells (MDSCs) and causes the dysfunction of tumor-reactive T cells. Here, we present a protocol for detecting ARG1 expression in tumor MDSCs from a murine model of colon cancer using flow cytometry. We describe steps for tumor tissue processing, antibody staining, and data acquisition. We then detail procedures for identifying MDSC subsets and detecting ARG1 expression using a precise gating strategy. For complete details on the use and execution of this protocol, please refer to Zhang et al.1.

Isolation, expansion, and adoptive transfer of human ILC2s for the treatment of mice bearing liquid and solid tumors.

Type 2 innate lymphoid cells (ILC2s) are crucial in regulating immune responses and various physiological processes, including tissue repair, metabolic homeostasis, inflammation, and cancer surveillance. Here, we present a protocol that outlines the isolation, expansion, and adoptive transfer of human ILC2s from peripheral blood mononuclear cells for an in vivo lineage tracking experiment in a mouse model. Additionally, we detail the steps involved in the adoptive transfer of human ILC2s to recipient mice bearing human liquid or solid tumors. For complete details on the use and execution of this protocol, please refer to Li et al.1.

Protocol to identify and isolate rare murine tumor-resident dendritic cell populations for low-input transcriptomic profiling.

Conventional type 1 dendritic cells (cDC1s) are critical for innate sensing of cancer, yet they are scarce in the tumor microenvironment (TME). Here, we present a protocol to identify and isolate cDC1 subsets from murine implantable tumors for subsequent transcriptomic profiling using a flow sorting-based strategy. We describe steps for cell culture of mouse tumors, tumoral growth, dissociation and isolation of tumoral cells, extracellular staining, and cell sorting. We then detail procedures for RNA isolation, mRNA library preparation, and sequencing. For complete details on the use and execution of this protocol, please refer to Papadas et al.1.

Protocol for establishing spontaneous metastasis in mice using a subcutaneous tumor model.

Recapitulating spontaneous metastasis in preclinical models is crucial for understanding mechanisms underlying cancer progression and testing effective therapeutic interventions. We present a protocol for establishing and characterizing the spontaneous metastasis model in mice. We describe steps for generating primary tumors, tumor resection, monitoring metastatic dissemination, and evaluating metastatic burden using histological and imaging techniques. This protocol provides a valuable tool for studying metastasis in vivo and testing therapeutic strategies aimed at preventing or targeting metastatic diseases. For complete details on the use and execution of this protocol, please refer to Liu et al.1.

Protocol for measuring the effects of an inhibitory signal associated with danger on honey bee dopamine levels.

The stop signal is produced in response to negative experiences at the food source and inhibits honey bee (Apis mellifera) waggle dancing. Here, we present a protocol for measuring the effects of an inhibitory signal associated with danger on honey bee dopamine levels. We describe steps for observing honey bee colonies, training them with artificial nectar, and simulating hornet attacks. We then detail procedures for recording waggle dancing and stop signals and measuring brain dopamine levels during different treatments. For complete details on the use and execution of this protocol, please refer to Dong et al.1.

Protocol for producing a rat model of non-obese prediabetes using a mild hypercaloric diet approach.

Metabolic disease complications pose a significant health risk due to their early development, making their diagnosis and radical therapy a considerable challenge. Here, we present a protocol for producing a rat model of non-obese prediabetes characterized by hyperinsulinemia, normoglycemia, and normal body weight. We describe steps for inducing the model in Sprague-Dawley (SD) male and ovariectomized female rats by free feeding on a mild hypercaloric diet. This protocol offers a potential model of metabolically unhealthy lean individuals. For complete details on the use and execution of this protocol, please refer to Elkhatib et al.1 and Dwaib et al.2.

Protocol for the purification of replisomes from the Xenopus laevis egg extract system for single-particle cryo-EM analysis.

Here, we present a large-scale FLAG immunoprecipitation protocol to isolate large protein complexes driving DNA replication at replicating chromatin assembled in Xenopus laevis egg extract. We describe how to prepare demembranated sperm nuclei (DNA) and low-speed supernatant egg extract (LSS) and present detailed procedures for sample preparation and application onto grids for negative stain electron microscopy (NS-EM) and cryoelectron microscopy (cryo-EM). For complete details on the use and execution of this protocol, please refer to Cvetkovic et al.1.

MIPs: multi-locus intron polymorphisms in species identification and population genomics.

The study of species groups in which the presence of interspecific hybridization or introgression phenomena is known or suspected involves analysing shared bi-parentally inherited molecular markers. Current methods are based on different categories of markers among which the classical microsatellites or the more recent genome wide approaches for the analyses of thousands of SNPs or hundreds of microhaplotypes through high throughput sequencing. Our approach utilizes intron-targeted amplicon sequencing to characterise multi-locus intron polymorphisms (MIPs) and assess genetic diversity. These highly variable intron regions, combined with inter-specific transferable loci, serve as powerful multiple-SNP markers potentially suitable for various applications, from species and hybrid identification to population comparisons, without prior species knowledge. We developed the first panel of MIPs highly transferable across fish genomes, effectively distinguishing between species, even those closely related, and populations with different structures. MIPs offer versatile, hypervariable nuclear markers and promise to be especially useful when multiple nuclear loci must be genotyped across different species, such as for the monitoring of interspecific hybridization. Moreover, the relatively long sequences obtained ease the development of single-locus PCR-based diagnostic markers. This method, here demonstrated in teleost fishes, can be readily applied to other taxa, unlocking a new source of genetic variation.

A simplified protocol for deriving sterile, infectious murine Heligmosomoides polygyrus bakeri larvae.

Gastrointestinal helminth infection occurs within a diverse microbiome, complicating the interpretation of whether effects are caused by the parasite versus the microbial community. Here, we present a protocol for deriving sterile larvae of the murine helminth, Heligmosomoides polygyrus bakeri (H. polygyrus), providing experimental control of the microbiome. We describe steps for sterilizing with a bleach solution and developing into infectious larvae using E. coli. We then detail procedures for removing bacterial contaminants before harvesting to ensure the generation of germ-free larvae.

Protocol for the characterization of the pancreatic tumor microenvironment using organoid-derived mouse models and single-nuclei RNA sequencing.

Single-nuclei RNA sequencing (snRNA-seq) allows for obtaining gene expression profiles from frozen or hard-to-dissociate tissues at the single-nuclei level. Here, we describe a protocol to obtain snRNA-seq data of pancreatic tumors from orthotopically grafted organoid-derived mouse models. We provide details on the establishment of these mouse models, the isolation of single nuclei from pancreatic tumors, and the analysis of the snRNA-seq datasets. For complete details on the use and execution of this protocol, please refer to Mucciolo et al.1.

Protocol to evaluate rat and mouse cardiomyocyte proliferation in vitro and in vivo.

Here, we present a protocol for the quantitative assessment of rat and mouse cardiomyocyte proliferation both in vitro and in vivo. For the in vivo approach, we describe steps for the isolation of neonatal rat cardiomyocytes and the employment of various indicators to quantify cell proliferation. We then detail in vivo procedures that incorporate comprehensive assays and a genetic lineage tracing strategy to evaluate endogenous cardiomyocyte proliferation. This protocol can be modified to investigate other mammalian cardiomyocyte proliferation. For complete details on the use and execution of this protocol, please refer to Ji et al.1.