STAR Protocols最新文献

Transcranial magnetic stimulation (TMS) perturbs specific brain regions and, combined with electroencephalography (EEG), enables the assessment of activity within their connected networks. We present a resting-state TMS-EEG protocol, combined with a controlled experimental design, to assess changes in brain network activity during offline processing, following a behavioral task. We describe steps for experimental design planning, setup preparation, data collection, and analysis. This approach minimizes biases inherent to TMS-EEG, ensuring an accurate assessment of changes within the network. For complete details of the use and execution of this protocol, please refer to Bracco et al.¹.

Here, we present a protocol for a preclinical ex vivo platform combining experimental flexibility with preservation of the tumor microenvironment. We outline steps for isolating human peripheral blood mononuclear cells (PBMCs), preparing patient-derived precision-cut tumor slices (PCTSs), cryopreserving the samples, and setting up the co-culture system. We provide instructions for treatment applications, interactions, and analyzing therapy responses. By preserving tumor architecture and heterogeneity, this model is applicable for evaluating tumor characteristics, immune interactions, and treatment efficacy in translational cancer research.

The lack of an etiologic Parkinson's disease (PD) model in non-human primates has inhibited our understanding of the disease and the development of therapies. Here, we present a protocol for generating a PD model in Macaca fascicularis. We describe steps for localizing the substantia nigra and striatum using magnetic resonance imaging (MRI). We then detail a technique for the intracerebral administration of adeno-associated virus (AAV)-mediated overexpression of α-synuclein into the substantia nigra plus the injection of poly(ADP-ribose) (PAR) into the striatum. For complete details on the use and execution of this protocol, please refer to Liu et al.¹.

Lymph nodes regulate immunity and maintain fluid balance in health and disease. Here, we present a protocol that uses normothermic perfusion to sustain patient-derived lymph nodes ex vivo for up to 24 h to study their structure and function. We describe steps for setting up both thermoregulatory and perfusion circuits, cannulating human lymph nodes, and perfusion. This protocol can be used to study how human lymph nodes change in cancer and other diseases, and/or in response to perturbations, including drugs. For complete details on the use and execution of this protocol, please refer to Barrow-McGee et al.¹.

Gene knockdown by small interfering RNA (siRNA) transfection is widely used to investigate gene function. Here, we present a protocol for the knockdown of the odorant-binding protein 2A (OBP2A) gene in a three-dimensional human epidermal equivalent model (3DE-model). We describe steps for growing human epidermal keratinocytes (normal human epithelial keratinocytes [NHEKs]) for 3DE-model construction in monolayer culture. We then detail procedures for transfecting the cells with siRNA, followed by a second siRNA transfection during 3DE-model construction. The efficiency of gene knockdown is verified by qPCR and ELISA. For complete details on the use and execution of this protocol, please refer to Nakanishi et al.¹.

Lactic acid bacteria (LAB) are a large group produced during carbohydrate fermentation, resulting in lactic acid. Here, we present a protocol for probiotic adhesion and interaction into buccal epithelial cells, chicken epithelial cells, and HT-29 cells, as well as for autoaggregation and coaggregation. We describe steps for visualizing LAB adhesion by imaging cell adhesion using a light microscope. We then detail procedures for performing an adhesion assay. For complete details on the use and execution of this protocol, please refer to Kumari et al.¹.

Plant m⁶A modification is highly dynamic, with plant hormones serving as key regulators that induce these dynamics. Here, we present a protocol for detecting phytohormone-induced changes in m⁶A modification using RNA dot blot and m⁶A sequencing (m⁶A-seq) techniques. We describe steps for the auxin treatment, RNA dot blot and m⁶A-seq library preparation, and data analysis. This protocol holds potential applications for analyzing changes in m⁶A modification induced by plant hormones. For complete details on the use and execution of this protocol, please refer to Bin et al.¹.

Spatially resolved transcriptomics (SRT) data contain intricate noise due to the diffusion of transcripts caused by tissue fixation, permeabilization, and cell lysis during the experiment. Here, we present a protocol for denoising SRT data using SpotGF, an optimal transport-based gene filtering algorithm, without modifying the raw gene expression. We describe steps for data preparation, SpotGF score calculation, filtering threshold determination, denoised data generation, and visualization. Our protocol enhances SRT quality and improves the performance of downstream analyses. For complete details on the use and execution of this protocol, please refer to Du et al.¹.

Here, we present a protocol for the quantitative characterization of human T cell aging. We describe steps for sample collection; peripheral blood mononuclear cell (PBMC) isolation; and the enrichment, assessment, and activation of naive CD8⁺ T cells. We then detail procedures for supernatant collection and quantification using the absolute copy number of mitochondrial DNA (mtDNA) released into the supernatants from in-vitro-activated naive CD8⁺ T cells. We also apply this approach to predict the occurrence of lung adenocarcinoma in middle-aged populations. For complete details on the use and execution of this protocol, please refer to Jin et al.¹.

RNA 5-methylcytosine (m⁵C) is a widespread modification and plays a crucial role in gene expression regulation. Here, we present a protocol for transcriptome-wide m⁵C methylome profiling at base resolution using bisulfite-free m⁵C detection strategy enabled by ten-eleven translocation (TET)-assisted chemical labeling sequencing (m⁵C-TAC-seq). We detail steps for TET-assisted chemical labeling, library construction, and data analysis. m⁵C-TAC-seq enables accurate and robust m⁵C detection in various RNA species. For complete details on the use and execution of this protocol, please refer to Lu et al.¹.