STAR Protocols最新文献

As Purkinje cells of the cerebellum have a very fast firing rate, techniques with high temporal resolution are required to capture cerebellar physiology. Here, we present a protocol to record physiological signals in humans using cerebellar electroencephalography (cEEG). We describe steps for electrode placement and recording. We then detail solutions for dealing with potential muscle, ocular, and electrical artifacts. This protocol has applications in recording patients with cerebellar disorders such as essential tremor, cerebellar ataxia, and dystonia. For complete details on the use and execution of this protocol, please refer to Pan et al.,¹ Wong et al.,² and Wang et al.³.

Host response to environmental exposures such as pathogens and chemicals can include modifications to the epigenome and transcriptome. Improved signature discovery, including the identification of the agent and timing of exposure, has been enabled by advancements in assaying techniques to detect RNA expression, DNA base modifications, histone modifications, and chromatin accessibility. The interrogation of the epigenome and transcriptome cascade requires analyzing disparate datasets from multiple assay types, often at single-cell resolution, derived from the same biospecimen. However, there remains a paucity of rigorous quality control standards of those datasets that reflect quality assurance of the underlying assay. This guide outlines a comprehensive suite of metrics that can be used to ensure quality from 11 different epigenetics and transcriptomics assays. Recommended mitigative actions to address failed metrics are provided. The workflow presented aims to improve benchwork protocols and dataset quality to enable accurate discovery of exposure signatures.

Under pathological conditions, astrocytes can transfer mitochondria to neurons, where they exert neuroprotective effects. In this context, we present a protocol for capturing astrocytic mitochondria in neurons of adult mice using a two-photon microscope. We describe an approach for constructing a mouse model with combined labeling of astrocytic mitochondria and neurons. We then detail procedures for the preparation of a coverslip with a customized titanium ring and cranial window for two-photon microscopy scanning. For complete details on the use and execution of this protocol, please refer to Zhou et al. ¹.

Microscopic cell segmentation typically requires complex imaging, staining, and computational steps to achieve acceptable consistency. Here, we describe a protocol for the high-fidelity segmentation of the nucleus and cytoplasm in cell culture and apply it to monitor interferon-induced signal transducer and activator of transcription (STAT) signaling. We provide guidelines for sample preparation, image acquisition, and segmentation. The approach performs indistinguishably from neural-network-based segmentation while requiring only conventional and cost-effective techniques. The protocol can be adapted to other signaling molecules undergoing nucleo-cytoplasmic shuttling and to high-throughput applications. This protocol enables simultaneous monitoring of two STAT isoforms using only conventional techniques and equipment and improves upon the assay published in Szanda et al.¹.

Blood cell aggregates are clinically useful biomarkers in a number of medical disorders. This protocol provides accurate and quantitative analysis of cell aggregates using a small volume of whole blood and imaging flow cytometry. We describe steps for sample collection, staining, and measurement. We then detail gating procedures and analysis of cell morphology. Sample preparation artifacts, activation, and morphological changes of cells are mitigated by omitting erythrocyte lysis and leukocyte isolation while maintaining high-throughput accurate imaging of leukocytes and platelets.

Alternating bilateral sensory stimulation (ABS) is a clinical physical therapy technique effective in treating post-traumatic stress disorder (PTSD). However, its utilization in treating conditions beyond PTSD remains limited. Here, we present a protocol to reduce ethanol-induced conditioned place preference (CPP) using 4 Hz ABS. We describe steps for the assembly of ABS apparatus, CPP training, and ABS treatment. This protocol has been validated in male mice and can be used to explore the mechanism behind the therapeutic effect of ABS. For complete details on the use and execution of this protocol, please refer to Lei et al.¹.

The intestine features a two-front nutrient supply environment, comprising an enteral side enriched with microbial and dietary metabolites and a serosal side supplied by systemic nutrients, collectively supporting intestinal and systemic homeostasis, but there is currently no optimal approach for extracting and assessing the local intestinal microenvironment. Here, we present a protocol for constructing a nutrient supply model in mice and extracting gut interstitial fluid (GIF) via centrifugation. This model and the extracted GIF are suitable for downstream analyses. For complete details on the use and execution of this protocol, please refer to Zhang et al.¹.

Hematopoietic stem cells (HSCs) generate blood and immune cells. Here, we present a protocol to differentiate human pluripotent stem cells (hPSCs) into hematopoietic progenitors that express the signature HSC transcription factors HLF, HOXA5, HOXA7, HOXA9, and HOXA10. hPSCs are dissociated, seeded, and then sequentially differentiated into posterior primitive streak, lateral mesoderm, artery endothelium, hemogenic endothelium, and hematopoietic progenitors through the sequential addition of defined, serum-free media. This 10-day protocol enables the manufacturing of blood and immune cells in monolayer cultures. For complete details on the use and execution of this protocol, please refer to Fowler and Zheng et al.¹.

Mammalian Dicer has been proved to be functional on double-stranded RNAs (dsRNAs) and involved in antiviral immunity or immune regulation. Here, we present a protocol for identifying Dicer as a dsRNA binding and cleaving factor to transfected dsRNA in cell lines, based on small RNA sequencing (RNA-seq) and dsRNA-immunoprecipitation (dsRNA-IP). We detail both experimental processes and analysis on small RNA-seq data. This protocol can be further applied to identify proteins that process dsRNA into small RNAs, like RNase L. For complete details on the use and execution of this protocol, please refer to Dai et al.¹.

Reduced expression of nucleolar genes induces stress and DNA damage. Here, we present a protocol to analyze DNA fragmentation at the single-cell level in Drosophila imaginal discs using an optimized alkaline comet assay. We describe steps for larvae development, tissue disaggregation, and single-cell dissociation. We then detail procedures for cell lysis, electrophoresis, and DNA visualization. This approach provides insights into the molecular consequences of nucleolar stress induction and its role in the DNA damage response pathways in vivo.