STAR Protocols最新文献

Humanized immune system (HIS) mouse models are commonly used for HIV studies; however, there is a need for sex-based data analysis. Here, we present a protocol to screen human donors for the CCR5delta32 (Δ32) polymorphism, which confers resistance to HIV infection, and identify the sex of the human donor used to produce HIS mice. We describe steps for human cell isolation from multiple tissue sources followed by genomic DNA isolation. We then detail PCR reactions and procedures for data analysis.

Here, we present a protocol for studying the neural mechanism of music therapy in alleviating depression using a combined electroencephalogram (EEG) and local field potential (LFP) signal recording. We describe steps for subject recruitment, surgery for implanting recording devices, signal processing, and emotional assessments. We then detail procedures for recording and analyzing EEG and LFP data. This protocol focuses on understanding how personal music preferences contribute to the therapeutic benefits of music in treating psychiatric conditions. For complete details on the use and execution of this protocol, please refer to Lv et al.¹.

To detect multiple protein markers, here we present a protocol for imaging pulmonary tuberculosis (PTB) lesions from murine lungs using Opal-tyramide signal amplification (TSA) conjugated fluorescent dyes in free-floating formalin-fixed thick lung sections (50-100 μm). We describe steps for preparing tissue sections, permeabilization and antigen retrieval, and multiplexing with antibodies raised in the same species. This protocol has been optimized to preserve tissue integrity and endogenously expressed fluorescent reporter signals and nuclear staining. It enhances the signal-to-background ratio and enables 3D image rendering.

Interstitial cells of Cajal (ICCs), pacemaker and neuromodulator cells in the gastrointestinal (GI) tract, play an important role in GI motility. However, quantifying ICCs is challenging due to their mixed morphologies. Here, we present a protocol for preparing and immunostaining ICC in the murine gastric tunica muscularis using artificial intelligence (AI). We describe steps for obtaining muscles, whole-mount staining, and imaging ICC using confocal microscope. We then detail procedures for training an AI to identify ICCs and quantify their volume. For complete details on the use and execution of this protocol, please refer to Taheri et al.¹.

We present a protocol to remove polyvinyl chloride-based microplastics (MPs) from aquatic environments using jute stick activated charcoal (JSAC) as an adsorbent. We describe steps for preparing JSAC by pyrolyzing jute sticks at high temperatures without oxygen, followed by chemical activation with HCl. We also detail the procedures for the post-functionalization of JSAC and for the collection, processing, and measurement of MPs. For complete details on the use and execution of this protocol, please refer to Alom et al.¹.

Intersample cellular heterogeneity (ISCH) is one of the largest contributors to DNA methylation (DNAme) variability. It is imperative to account for ISCH to accurately interpret analysis results in epigenome-wide association studies. We compiled this primer based on the current literature to guide researchers through the process of estimating and accounting for ISCH in DNA methylation studies. This primer outlines the procedure of bioinformatic ISCH prediction, including using reference-based and reference-free algorithms. It then follows with descriptions of several methods to account for ISCH in downstream analyses, including robust linear regression and principal-component-analysis-based adjustments. Finally, we outlined three methods for estimating differential DNAme signals in a cell-type-specific manner. Throughout the primer, we provided statistical and biological justification for our recommendations, as well as R code examples for ease of implementation.

Many widely used spatial transcriptomics technologies, such as Visium, capture data at multicellular resolution, precluding single-cell analysis. Here, we present scResolve, a computational protocol to recover single-cell gene expression profiles from low-resolution spatial transcriptomics data. We describe steps for computational environment setup and preparing data and formatting. We then detail procedures for running super-resolution inference and cell segmentation modules. scResolve runs in a cluster environment, leveraging parallel computing to accelerate data processing and deliver faster results. For complete details on the use and execution of this protocol, please refer to Chen et al.¹.

We introduce a protocol for generating 3D organoids from circulating tumor cells (CTCs), enabling longitudinal functional and molecular analyses in pancreatic cancer patients, including those with unresectable disease, which constitutes the majority of cases. We outline the process for isolating and characterizing CTCs from the blood of pancreatic cancer patients and provide detailed instructions for initiating, passaging, and phenotyping CTC-derived organoids. Additionally, we describe techniques for utilizing these organoids in drug screening with a focus on stemness-related pathways. For complete details on the use and execution of this protocol, please refer to Tang et al.¹.

The open-field tank test is a well-validated technique for evaluating anxiety behavior in vertebrate animal models. However, manual behavioral analysis can be subjective and time consuming. Here, we present a protocol using ZebraTrack, an open-source ImageJ-based approach for distraction-free swimming video acquisition and automated tracking of adult zebrafish. We detail steps for mathematical calculations on 2D coordinates to extract various behavioral endpoints for anxiety-behavioral evaluation. For complete details on the use and execution of this protocol, please refer to Nema et al.¹.

Protein UFMylation regulates numerous cellular processes including ribosome quality control and nuclear DNA repair. Here, we present a technique to isolate nuclei and purify UFMylated proteins under denaturing non-reducing conditions from commonly used mammalian cell line models such as hTERT-RPE1, HEK293, U2OS, and HCT116 cells. We then describe procedures for identifying and analyzing purified UFMylated proteins using mass spectrometry and western blot. For complete details on the use and execution of this protocol, please refer to Panichnantakul et al.¹.