Ramseywoods4326

Bumblebees are a key pollinator. Understanding the factors that influence the timing of sleep and foraging trips is important for efficient foraging and pollination. Here, we illustrate how individual locomotor activity monitoring and colony-wide radio frequency identification tracking can be combined to analyze the effects of agrochemicals like neonicotinoids on locomotor and foraging rhythmicity and sleep quantity/quality in bumblebees. We also highlight aspects of the design that can be adapted for other invertebrates or agrochemicals, allowing broader application of these techniques. For complete details on the use and execution of this protocol, please refer to Tasman et al. (2020).Here, we describe protocols for the preparation and dissociation of human fetal and pediatric intestinal tissue to a high-viability epithelial single-cell suspension. This epithelium-enriched single-cell suspension can then be used to generate single-cell RNA sequencing data as well as to create human intestinal organoids from both the fetal and pediatric intestine. Finally, this protocol details the dissociation of the intestinal organoids for use in single-cell analysis or passaging of organoids. For complete details on the use and execution of this protocol, please refer to Elmentaite et al. (2020).During adulthood, the activation of adult neural stem cells (NSCs) has been mostly studied ex vivo in post-mortem tissues or in vivo in anesthetized animals. This protocol presents an approach that allows for the long-term and minimally invasive investigation of adult NSC activation and physiology in freely behaving animals. By combining specific NSC labeling and mini-endoscopic microscopy, live imaging of NSC division and Ca2+ activity can be performed continuously for 2-3 days and even up to several months. For complete details on the use and execution of this protocol, please refer to Gengatharan et al. (2021).Anti-PD-1/PD-L1 therapy shows long-term effects in many cancer types, but resistance and relapse remain the main limitations of this therapy. Here, we describe a protocol to evaluate the tumor response to immunotherapy in a mouse lung cancer model. The protocol includes the establishment of the lung cancer mouse model, anti-PD-1 treatment, tumor-infiltrating lymphocyte isolation, immunofluorescence, and flow cytometry analysis. This protocol can also be applied to other cancer types and immunotherapies. For complete details on the use and execution of this protocol, please refer to Yu et al. (2021).Organoid models have been shown to be valuable tools for studying epithelial-mesenchymal crosstalk during biological and pathological settings. Our data identified ACTA2+ PDGFRα+ repair-supportive mesenchymal cells as an important component of the conducting airway niche. Here, we provide a detailed protocol for culturing airway organoids, or bronchiolospheres, which provide an assessment of the ability of mesenchymal cells to support club-cell growth. For complete details on the use and execution of this protocol, please refer to Moiseenko et al. (2020).Here, we describe a protocol for a photoaffinity labeling probe strategy for target deconvolution in live cells. We made a chemical probe by incorporation of a photoreactive group to covalently cross-link with adjacent amino acid residues upon UV irradiation. Click chemistry-based enrichment captures labeled proteins for proteomic analysis. Here, we detail specifics for finding targets of LXRβ, but the protocol has potential for application to other targets. For complete details on the use and execution of this protocol, please refer to Seneviratne et al. (2020).We have outlined the approach of visualizing autophagy specifically in the epithelial follicle stem cells of the Drosophila ovary using the LysoTracker dye. The advantage of using this protocol is that it details several techniques, including ovary dissection, immunofluorescence, and western blotting, that positively identify autophagy changes in a very small population of cells. One of the limitations of this protocol is that it needs to be combined with other genetic manipulations and positive markers of the autophagy pathway. For complete details on the use and execution of this protocol, please refer to Singh et al., (2018).Here, we describe a rapid and versatile protocol to generate gapped DNA substrates for single-molecule (SM) analysis using optical tweezers via site-specific Cas9 nicking and force-induced melting. learn more We provide examples of single-stranded (ss) DNA gaps of different length and position. We outline protocols to visualize these substrates by replication protein A-enhanced Green Fluorescent Protein (RPA-eGFP) and SYTOX Orange staining using commercially available optical tweezers (C-TRAP). Finally, we demonstrate the utility of these substrates for SM analysis of bidirectional growth of RAD-51-ssDNA filaments. For complete details on the use and execution of this protocol, please refer to Belan et al. (2021).The cooperativity of six cations (Ca2+, Mg2+, Zn2+, Al3+, Cr3+ and Fe3+), three pectins (sugar beet, high and low methyl esterified), three dispersed phases (medium chain triglycerides (MCT), orange oil and hexadecane), time (30 days) and pH (2.0 and 6.0) has been investigated in the formation and stability against coarsening of oil-in-water emulsions. Cations generally influenced emulsion stability in the following order (most stable) Ca2+ > Mg2+ > Al3+ > Cr3+ > Zn2+ > Fe3+ (least stable). This order largely coincided with that of the strength of pectin-cation interactions showing that the higher the affinity of cation for pectin the less stable the emulsion. More stable emulsions were formed with sugar beet pectin, which was also unresponsive to the presence of cations, followed by high- and then low-methyl esterified samples. At pH 2.0 all pectins showed their best emulsification performance whereas shifting pH to 6.0 severely impaired emulsification capacity and longer term stability against droplet growth. Smaller droplets were created with hexadecane under all conditions studied followed by MCT and orange oil in agreement with their aqueous solubilities. The present results advance our understanding of the stabilisation of emulsions using pectin and allow us to tailor their functionality for applications in food, pharmaceutical and biomedical industries.