STAR Protocols最新文献

The brain, specifically the hypothalamus, is a key regulator of the homeostatic balance of numerous biomolecules in the body via its control of the neuroendocrine system. Here, we present a protocol to study body vitamin A balance by injecting synthetic retinoids into the third ventricle of the rat brain. We also describe steps for the intraventricular application of viral vectors to the arcuate nucleus of a mouse brain to knock down vitamin A homeostatic genes. This approach is applicable to the study of vitamin A homeostasis. For complete details on the use and execution of this protocol, please refer to Imoesi et al.¹.

Complementary metal-oxide-semiconductor (CMOS)-based high-density microelectrode arrays (HD-MEAs) enable neuronal recordings with high spatiotemporal resolution. However, integrating polydimethylsiloxane (PDMS) microstructures onto HD-MEA surfaces to control network architecture is currently challenging and platform specific. Here, we present a protocol for PDMS fabrication, HD-MEA chip preparation, PDMS-HD-MEA microstructure alignment, and cell culture, including alternatives. Our results show reproducible formation of modular networks with characteristic activity patterns across different systems. This protocol supports engineering of defined neuronal architectures while maintaining compatibility with various HD-MEA systems. For complete details on the use and execution of this protocol, please refer to Sato et al.¹.

Nuclear-enriched long non-coding RNAs (lncRNAs) can regulate gene expression by interacting with specific RNA-binding proteins (RBPs). Here, we present a protocol to directly image these lncRNAs at the single-molecule level together with their protein interactors in human cardiomyocytes. We describe steps to perform RNA-fluorescence in situ hybridisation (FISH) of a nuclear intron-retaining lncRNA, followed by sequential protein immunofluorescence (IF), enabling simultaneous visualisation of specific RNA and protein targets. We also detail procedures for probe design, cell seeding, and fixation. For complete details on the use and execution of this protocol, please refer to Buonaiuto et al.¹.

In plants, differentiation and expansion of some cell types is accompanied by exit from cell division and onset of endoreplication. Here, we describe a protocol to study changes in mRNA levels during endoreplication in Arabidopsis thaliana cotyledons. We provide detailed steps for the isolation of total nuclei and a strategy to sort stained nuclei according to their ploidy level. Finally, we detail procedures for RNA extraction, a housekeeping gene, and a marker gene for qPCR across ploidy.

Here, we present a protocol for the small-molecule-driven derivation of dopaminergic and GABAergic neurons from neural progenitor cells (NPCs) differentiated from human induced pluripotent stem cells and for stereotactic xenotransplantation of NPCs into the mouse forebrain. We also describe steps for perfusion of xenografted mice and microdissection of grafts for single-nucleus RNA sequencing analysis. This protocol can be used for various assays, including drug screening and toxicity assays, or for mechanistic studies on human neurons. For complete details on the use and execution of this protocol, please refer to Reinhardt et al.¹ and Al-Dalahmah et al.².

Transmembrane proteins are notoriously difficult to isolate from tissue samples due to their hydrophobic nature and often low abundance. Here, we describe a protocol for the isolation, enrichment, and detection of the high-molecular-weight adhesion-type G protein-coupled receptor (aGPCR) member ADGRL/Latrophilin/Cirl from lipid-rich Drosophila melanogaster pupae. We detail steps to collect and homogenize pupae, enrich Cirl through immunoprecipitation, and visualize protein bands after SDS-PAGE and Western Blot. This protocol can be readily adapted to other tissues and (transmembrane) proteins. For complete details on the use and execution of this protocol, please refer to Bormann et al.¹.

Here, we present a protocol for estimating biomass and carbon sequestration in tropical rainforests. We describe steps for combining remotely sensed data from Sentinel-2 imagery with data collected from standard field surveys. This protocol provides a cost-effective and time-efficient solution for large-scale forest resource assessments. For complete details on the use and execution of this protocol, please refer to Dang et al.¹.

Here, we present a protocol to investigate the mechanism and regulation of human mitochondrial transcription initiation by integrating biochemical and cryoelectron microscopy (cryo-EM) approaches. We describe the steps for expressing and purifying POLRMT, TFB2M, and TFAM; assembling transcription initiation complexes; and utilizing single-particle cryo-EM analysis for the high-resolution structural determination of transcription intermediates. We provide assays for quantifying promoter melting, ATP binding, abortive RNA synthesis, and RNA slippage steps, enabling structure-function studies and gaining mechanistic insights into transcription initiation and transcription factor regulation. For complete details on the use and execution of this protocol, please refer to Shen et al.¹.

Itaconate mimetics inadequately represent endogenous itaconate, a negative regulator of innate immune-driven pro-inflammatory cytokines. We present a CRISPR-Cas9 protocol to delete a 4-nucleotide region in the immunoresponsive gene 1 (IRG1), ablating ACOD1 (the itaconate-producing enzyme) in THP-1 cells. We describe the functional validation of ACOD1 deletion using immunoblotting, ELISA, and liquid chromatography-mass spectrometry (LC-MS) quantification of itaconate, enabling the study of endogenous itaconate in THP-1 macrophages. For complete details on the use and execution of this protocol, please refer to Bourner et al.¹.

Functional genomic studies rely on controlled gene expression, which is challenging for essential genes or those active only under specific conditions. We provide a protocol for generating Magnaporthe oryzae (synonym of Pyricularia oryzae) with conditional BUF1 gene expression. We describe steps for using targeted promoter replacement to substitute the native BUF1 promoter with the nitrogen-responsive promoter. This approach enables nitrogen source-dependent control of expression in M. oryzae, leading to observable pigmentation changes that facilitate easy phenotypic screening.