STAR Protocols最新文献

Dual spatial transcriptomics analysis offers profiling of host- and pathogen-specific transcriptional patterns in infected tissues to establish pathoadaptation signatures and predict infection outcomes. Here, we present a protocol for tissue processing, imaging, selection of regions of interest, library preparation, sequencing, and analysis pipeline. This protocol has a potential application for the analysis of any infected tissue. For complete details on the use and execution of this protocol, please refer to Zhou et al.¹.

Available mouse models for pancreatic ductal adenocarcinoma (PDAC) are limited by slow tumor development and failure to recapitulate key stromal and immune characteristics. Here, we present a protocol for generating a collagen hydrogel mouse model for orthotopic PDAC. We describe steps for embedding mouse pancreatic cancer cells in a dense collagen hydrogel and surgically implanting it into the mouse pancreas. Mouse PDAC tumors typically reach 1 cm in diameter by 10 days after implantation and show immune and stromal cell recruitment. For complete details on the use and execution of this protocol, please refer to Korah et al.¹.

CD70 has emerged as a promising immunotherapeutic target in renal cell carcinoma (RCC), with both CD70-directed monoclonal antibodies and chimeric antigen receptor (CAR)-based therapies currently under development. Here, we describe a protocol for the generation of human CD70-directed allogeneic CAR-natural killer T (NKT) (^AlloCAR70-NKT) cells derived from cord blood CD34⁺ hematopoietic stem and progenitor cells (HSPCs) using a clinically guided culture. Furthermore, we describe the therapeutic efficacy of ^AlloCAR70-NKT cells in mediating cytotoxic activity against RCC cell lines in vitro. For complete details on the use and execution of this protocol, please refer to Li et al.¹.

Here, we present an optimized high-resolution Hi-C protocol for human breast tissues using the Phase Genomics Proximo Hi-C Kit. We describe steps for liquid nitrogen tissue processing, crosslinking, and quenching, followed by cell lysis and controlled chromatin fragmentation. Proximity ligation captures three-dimensional interactions, followed by reverse crosslinking, DNA purification, and streptavidin bead enrichment. Libraries are prepared on beads, amplified, and cleaned with size selection, producing high-quality material for Illumina sequencing and genome-wide 3D chromatin analysis. For complete details on the use and execution of this protocol, please refer to Choppavarapu et al.¹.

Here, we present a protocol for synthesizing and purifying high-yield, short-length poly(ADP)ribose (PAR) polymers using fast protein liquid chromatography (FPLC). We describe steps for expressing and purifying proteins, in vitro synthesis of PAR, and fractionation and visualization of PAR. This cost-effective, non-hazardous, shorter approach avoids the use of radiolabeled substrates, hazardous reagents, and specialized equipment, producing homogenous PAR chains under 10 units. It eliminates the need for enzymes such as PARG and SVP, enabling broad accessibility for biophysical and structural studies.

Protein citrullination occurs through the deimination of peptidyl-arginine to yield peptidyl-citrulline by one of the peptidyl-arginine deiminase (PAD) family members. This protocol identifies citrullinated protein residues using immunoprecipitation followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We describe steps for identification of citrullination modifications in vitro or in cell culture, immunoprecipitation of nuclear citrullinated proteins, and identification of citrullinated residues by mass spectrometry (MS). This protocol applies to both recombinant protein assays and in vitro cell culture assays. For complete details on the use and execution of this protocol, please refer to Indeglia et al.¹.

Brown adipose tissue (BAT) represents a specialized form of adipose tissue that has attracted much attention in the search for therapeutic interventions to treat obesity and its related metabolic disorders. Here, we present a protocol to modulate BAT metabolism in vivo in mice via the direct administration of adenovirus (Ad). We also describe steps for the delivery of postoperative care, including the administration of analgesics and the application of antiseptics to the surgical site to prevent potential infections. For complete details on the use and execution of this protocol, please refer to Al-Massadi et al.¹.

Murine cerebral organoids provide a rapid and reproducible in vitro system that recapitulates key aspects of neurogenesis. While human cerebral organoid protocols are well established, methods for non-human models remain limited. Here, we present a protocol for generating long-term cultured murine cerebral organoids from E14.5 embryonic stem cells (ESCs). We describe steps for generating mature organoids, followed by histological processing including paraffin embedding and microtome sectioning. We then detail procedures for characterizing murine cerebral organoids through H&E staining and immunofluorescence techniques.

Here, we present a protocol for generating and selecting stable transgenic tomato lines through Agrobacterium tumefaciens-mediated transformation of cotyledon and hypocotyl explants. We describe steps for sterilizing and planting tomato seeds, cotyledon and hypocotyl excision and preculture, Agrobacterium tumefaciens preparation and co-cultivation with explants, and recovering explants and bacterial overgrowth restriction. We then detail procedures for selecting green calli; regenerating explant excision and growth in non-selective media; plant hardening, potting, and growing; and T-DNA insertion confirmation by PCR. For complete details on the use and execution of this protocol, please refer to Mountourakis et al.^1,2,3.

Fibro-adipogenic progenitors (FAPs) are key regulators of skeletal muscle regeneration and influence myogenic differentiation. Here, we present a protocol for the isolation of primary FAPs from injured murine skeletal muscle and the co-culture of C2C12 myoblasts with either 3T3-L1 or primary FAPs. We describe steps for muscle injury, harvest, and digestion followed by FAP isolation. We then detail procedures for co-culture and for assessing myogenic differentiation using immunofluorescence imaging, enabling direct comparison of stromal influences on myoblast differentiation. For complete details on the use and execution of this protocol, please refer to Norris et al.¹.