STAR Protocols最新文献

In vitro models of neuronal aging and gene manipulation in human neurons (hNeurons) are valuable tools for investigating human brain aging and diseases. Here, we present a protocol for applying human embryonic stem cell (hESC)-derived neurons to model aging and the further application of small interfering RNA (siRNA)-mediated gene silencing for functional investigations. We describe steps for neuronal differentiation and culture, siRNA transfection, and technical considerations to ensure reproducibility. Our protocol enables investigations of the molecular mechanism underlying neuronal aging and facilitates drug evaluation. For complete details on the use and execution of this protocol, please refer to Zhang et al.¹.

Characterization of zebrafish mutants is routinely time consuming as morphological phenotypes can be subtle or variable, thus hampering robust quantification. Here, we present a protocol to identify the overall phenotype by overlaying images of lymphatic vessels acquired from multiple zebrafish embryos of a genetic background. We describe steps for preparing samples, obtaining confocal images, and their computational alignment. In addition, we supply sample images for testing. For complete details on the use and execution of this protocol, please refer to Arnold et al.¹.

Inhibitory synaptic transmission mediated by the neurotransmitter γ-aminobutyric acid (GABA) is dependent on the concentration of chloride ions (Cl^-) in neurons, which can be assessed by making patch-clamp recordings of the reversal potential for GABA (E_GABA). Here, we present a protocol to study the regulation of cation-chloride cotransporters and the strength of synaptic inhibition in cultured mouse cortical neurons using electrophysiology. We describe steps for culturing neurons isolated from postnatal pups and electrophysiological measurement of E_GABA. For complete details on the use and execution of this protocol, please refer to Raveendran et al.¹.

Human induced pluripotent stem cells (hiPSCs) can be used to generate assembloids that recreate thalamocortical circuitry displaying short-term and long-term synaptic plasticity. Here, we describe a protocol for differentiating hiPSCs into thalamic and cortical organoids and then fusing them to generate thalamocortical assembloids. We detail the steps for using whole-cell patch-clamp electrophysiology to investigate the properties of synaptic transmission and synaptic plasticity in this model system. For complete details on the use and execution of this protocol, please refer to Patton et al.¹.

Owing to inconsistencies in human B cell classification and the difficulty in distinguishing heterogeneous subpopulations, we present a protocol to construct gene regulatory networks and gene activity landscapes for human B cell developmental stages. We describe steps for acquiring bone marrow data; conducting single-cell downstream analysis; and leveraging the St. Jude Algorithm for the Reconstruction of Accurate Cellular Networks (SJARACNe), Network-based Bayesian Inference of Drivers (NetBID2), and single-cell Mutual Information-based Network Engineering Ranger (scMINER) algorithms for network-based analysis. Our protocol elucidates the biological characteristics of developmental stages in human B cells. For complete details on the use and execution of this protocol, please refer to Huang et al.¹.

Cap analysis of gene expression (CAGE) is a technique that facilitates the assessment of the 5'-end of RNA transcript starting site (TSS) of both coding and non-coding genes. Here, we present a protocol for using CAGE on Illumina patterned flow cell technology with dual indexes on mouse and human samples. We describe steps for sequencing, automated data processing, and complete analytical framework ensuring CAGE operability for the determination of TSS and enhancers on the ever-evolving Illumina sequencing platforms.

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.

Endothelial cells (ECs) play a major role in vascular homeostasis and pathology. They show a pronounced heterogeneity between different organs as well as within the same organ. Here, we present a protocol for the rapid site-specific isolation of murine aortic ECs. We describe steps for vessel preparation and the isolation of EC layers by freezing on glass coverslips using a modified Häutchen technique. This protocol can be adapted to investigate surface cells from other tissues as well. For complete details on the use and execution of this protocol, please refer to Brückner 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 medial entorhinal cortex (MEC) is crucial for context memory, yet its role in context-induced retrieval of morphine withdrawal memory remains to be investigated. Here, we present a protocol to evaluate the importance of projection neurons from the MEC to the basolateral amygdala (BLA) (MEC^-BLA neurons) in mice during context-induced retrieval of morphine withdrawal memory using a conditioned place aversion (CPA) model. We describe steps for surgical procedure and behavioral experiments. Then, we detail procedures of immunofluorescence staining and image analysis. For complete details on the use and execution of this protocol, please refer to Fu et al.¹.