STAR Protocols最新文献

Notch signaling is a pivotal regulator in the vascular system that is essential for development, angiogenesis, and maintaining vascular homeostasis. Here, we present a protocol for tyramide signal amplification (TSA) immunohistochemistry, tailored explicitly for detecting Notch signaling components in vascular tissues. We describe steps for utilizing tailored antigen retrieval techniques, specific blocking solutions, and a complex of avidin/biotin-horseradish peroxidase conjugate with tyramide, along with optimized washing steps. For complete details on the use and execution of this protocol, please refer to Zhu et al.¹.

cfos is an immediate early gene commonly used to identify neuronal activation. After loud sound stimulation, neurons in the inferior colliculus are activated and cFos is expressed in the nucleus. Here, we present a protocol for quantifying neuronal activity in response to auditory stimulation using cFos immunostaining in the mouse inferior colliculus. We then detail procedures for image acquisition and analysis. For complete details on the use and execution of this protocol, please refer to Louros et al.¹.

T cell responses play an important role in viral clearance and prevention of infection. Here, we present a protocol to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell responses using spectral flow cytometry. We describe steps for peripheral blood mononuclear cell (PBMC) thawing and stimulation with SARS-CoV-2 spike peptide pools. We then describe detailed procedures for cell confining, staining, and fixation. This protocol has potential application in spectral flow cytometry on other virus-specific T cell responses. For complete details on the use and execution of this protocol, please refer to Zhao et al.¹.

Here, we present a protocol for generating the lung cancer cell line, LLC1-BMT5, with highly brain metastatic tropism through multiple rounds of in vivo selection. We describe steps for establishing the brain metastases (BrMs) mouse model through intracardiac injection of cancer cells. We then detail procedures for obtaining brain metastatic subpopulations, in vivo selection of LLC1-BMT5 cells, and validating metastatic potential. This protocol will facilitate the study of the molecular mechanisms of BrMs and the development of anti-BrM treatment strategies. For complete details on the use and execution of this protocol, please refer to Wang et al.¹.

The interoperability of variant identification pipelines is fundamental for achieving consistent clinical care across oncology research centers and hospitals. Here, we present a protocol for using ONCOLINER, a platform for the assessment, improvement, and harmonization of somatic variant discovery of multiple pipelines. We describe steps for acquiring benchmarking datasets and executing the user variant calling pipeline. We then detail the procedures for performing analyses to produce user-friendly reports showing the quality, scope, and applicable improvements for each tumor genome analysis. For complete details on the use and execution of this protocol, please refer to Martín et al.¹.

Conventional dendritic cells (cDC) are professional antigen-presenting cells able to prime naive T cells. Here, we present a protocol for ex vivo T cell priming by murine splenic cDC. We describe the steps of injecting fluorescently labeled antigens to mice, purifying antigen-bearing cDC, and priming antigen-specific T cells ex vivo. This protocol is suitable for studying the T cell priming function of cDC in various murine models and helps factor in the effect of the microenvironment on cDC ability to uptake and process antigens. For complete details on the use and execution of this protocol, please refer to Ashayeripanah et al.¹.

Here, we present a protocol for monitoring phagocytosis by M2-type macrophages using automated counting of phagocytic events with an imaging cytometer. We describe steps for isolating and differentiating peripheral blood mononuclear cell (PBMC)-derived monocytes into M2-like macrophages, preparing cancer cells expressing a green fluorescence marker, labeling with a pH-sensitive dye, and co-culturing with macrophages. We then outline procedures for enumerating phagocytic events using an imaging cytometer. For complete details on the use and execution of this protocol, please refer to Mishra et al.¹.

Bioluminescence resonance energy transfer (BRET) allows to quantitate protein interactions in intact cells. Here, we present a protocol for measuring BRET due to transient interactions of oncogenic K-RasG12V in plasma membrane nanoclusters of HEK293-EBNA cells. We describe steps for seeding, transfecting, and replating cells. We then detail procedures for their preparation for BRET measurements on a CLARIOstar microplate reader and detailed data analysis. For complete details on the use and execution of this protocol, please refer to Steffen et al.¹.

Genome-wide association study loci likely influence disease risk through cis-regulation of nearby genes. We present a protocol for measuring the cis-regulatory activity of risk variants on specific gene promoters in human primary astrocytes using luciferase reporter assays. We describe steps for transfection, conducting the assay, and data analysis. The transfection could be adapted to enable validation of putative risk variants in any cell type. For complete details on the use and execution of this protocol, please refer to Littleton et al.¹.

The C. elegans nervous system is compact and well described, ideal for identifying genetic programs that drive neuron-specific development, function, and connectivity. Here, we present a protocol for isolating specific neuron types for gene expression profiling by bulk or single-cell RNA sequencing. We describe steps for worm synchronization, dissociation, and fluorescence-activated cell sorting (FACS) isolation. We then detail procedures for RNA extraction and preparing cells for single-cell sequencing. This protocol is applicable to the isolation of individual cell types from larval and adult animals. For additional details on the use and execution of this protocol, see Taylor et al.¹.