STAR Protocols最新文献

ONE vector G protein Optical (ONE-GO) biosensors can measure the activity of endogenously expressed G protein-coupled receptors (GPCRs) in primary cells. By detecting G proteins that belong to all four families (G_s, G_i/o, G_q/11, G_12/13) across cell types, these biosensors provide high experimental versatility. We first describe steps to express ONE-GO biosensors in primary cells using lentiviral transduction. We then detail how to carry out measurements and subsequent analysis to quantify changes in bioluminescence resonance energy transfer (BRET) reporting on endogenous GPCR activity. For complete details on the use and execution of this protocol, please refer to Janicot et al.¹.

Kaposi sarcoma (KS) punch biopsies present unique challenges for extracting nucleic acids, which can be exacerbated by their long-term stabilization in RNAlater. Here, we present a protocol for simultaneously isolating DNA, RNA, and miRNA from a single KS punch biopsy. We detail the steps for preparing reagents and supplies, disrupting KS tissue using manual and mechanical methods, isolating DNA and total RNA, evaluating nucleic acid quality, and storing nucleic acids long-term.

The study of reproductive function in turkey hens has been difficult due to the lack of a reliable, representative in vitro model for investigating profound physiological aspects. This article presents a protocol to establish turkey oviductal organoids, including steps for isolating turkey oviduct epithelial cells followed by seeding and maintaining 3D organoid cultures. We also detail procedures for organoid fixation for histological analysis. This organoid model could serve as a valuable in vitro tool for understanding the intricacies of turkey reproductive physiology.

Skeletal muscle spatial analyses have revealed unexpected regionalized gene expression patterns challenging the understanding of muscle as a homogeneous tissue. Here, we present a protocol for the spatial analysis of transcript and protein levels in murine skeletal muscle. We describe steps for tibialis anterior dissection, formaldehyde fixation, tissue chopper cutting, and hybridization chain reaction (HCR) detection and amplification. We then detail procedures for immunostaining, tissue clearing, and imaging. This protocol is easily adaptable to other tissues.

Invadopodia are actin-rich protrusions on the tumor cell membrane that degrade the extracellular matrix and play a crucial role in tumor cell invasion and metastasis. Here, we present a protocol to examine invadopodia's ability to form and degrade the extracellular matrix during tumor invasion and metastasis. We detail the procedure for using immunofluorescence staining to indirectly detect invadopodia formation and assess their extracellular matrix degradation capability via the gelatin degradation assay. For complete details on the use and execution of this protocol, please refer to Huang et al.¹.

Disclosure of the key events in tumor progress will help us deepen the understanding of tumorigenesis. Here, we present a protocol for multi-regional tissue capture of malignant continuum. We describe steps for preparing tissue sections, laser capture microdissection, and whole-genome library preparation, which enable the concurrent analysis of potential driver events in precancer initiation. This protocol overcomes challenges posed by limited DNA quantity and preserves the spatial information of the target regions. For complete details on the use and execution of this protocol, please refer to Li et al.,¹ Chang et al.,² and Chen et al.³.

Cancer cells with homologous recombination deficiency (HRD) exhibit a distinctive vulnerability to poly(ADP-ribose) polymerase inhibitors (PARPis). To address the limitations of existing methodologies incapable of providing real-time insights into homologous recombination (HR) status, we present an adenovirus-based functional assay designed to quantify cellular HR activity. Here, we delineate the step-by-step procedure for producing the adenovirus harboring an HR reporter, processing primary cells, and assessing HR activity in primary ovarian cancer cells. For complete details on the use and execution of this protocol, please refer to Lee et al.¹.

Complement proteins contribute to neurodegeneration in Alzheimer's disease (AD) and are secreted by glia surrounding beta-amyloid (Aβ) plaques. We present an optimized protocol for Aβ plaque detection with tyramide-digoxigenin signal amplification. This is combined with a multiplex mRNA fluorescence in situ hybridization (FISH) panel to assay glial-specific complement expression proximal to Aβ plaques in TauPS2APP mice. We describe steps for tissue preparation and mRNA detection. We then detail steps for the detection of Aβ plaques, image acquisition, and analysis.

The ribosome-associated protein quality control (RQC) core factor nuclear export mediator factor (NEMF) appends C-terminal extended sequences (CESs) to ribosome-stalled nascent chains (NCs). Specific CESs compositions could be directly recognized by enzymes and facilitate NC degradation. Yet, NEMF-mediated CESs remains largely unidentified. Here, we present a protocol for identifying and characterizing NEMF-mediated C-terminal modifications on mitochondrial NCs (mitoNCs) via tandem mass spectrometry (MS/MS) analysis. We describe strategies aimed at constructing a customized MS/MS spectra database for unknown CESs and detail the steps for CES-modified sample preparation. For complete details on the use and execution of this protocol, please refer to Lv et al.¹.

Isolation of antigen-specific plasma B cells had been challenging until the recent arrival of the Beacon platform. Leveraging light-sorting technology, Beacon can perform high-throughput screening of plasma B cells on a chip to sort single cells with the desired antigen specificity. Here, we present a protocol for isolating antigen-specific plasma B cells from immunized mice using Beacon, sequencing the encoded B cell receptors (BCRs), and cloning and expressing the resulting antibodies. This protocol can easily be extended to human samples.