University of Bristol

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

This protocol describes the stimulation of cultured fibroblasts with low-intensity pulsed ultrasound, which drives focal adhesion formation and Rac1 activation by mimicking engagement of the transmembrane matrix receptor, syndecan-4. This approach allows investigation of a successful clinical technique at the cellular level, thereby providing opportunities for refinement of the therapy.

The method to measure adult Drosophila associative memory is described. The assay is based on the ability of the fly to associate an odor presented with a negative reinforcer (electric shock) and then recall this information at a later time, allowing memory to be measured.

Here we describe a general method for random microseed matrix screening. This technique is shown to significantly increase the success rate of protein crystallization screening experiments, reduce the need for optimization, and provide a reliable supply of crystals for data collection and ligand-soaking experiments.

The chronically instrumented non-anesthetized fetal sheep model is used to study human fetal development in health and disease, because it permits surgical placement and maintenance of catheters and electrodes, repetitive blood sampling, substance injection, recording of bioelectrical activity, and in vivo imaging. We describe the procedures required to establish this model.

Here, we present a protocol to induce inflammatory arthritis in vivo using antigen response, termed Antigen-induced Arthritis (AIA), an effective tool for research into Rheumatoid Arthritis (RA) research. Additionally we demonstrate the therapeutic effects of infusion of MSC to reduce inflammation, cartilage degradation and expression of TNFα.

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

This protocol compares the relative affinities of binding partners for Rho-family GTPases, including Rac1. In vivo, Rac1-binding proteins compete for a single binding interface, the conformation of which is dictated by a bound nucleotide. The nucleotide is both important and difficult to control experimentally, due to the high hydrolysis rate.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

Finite Element Analysis is a frequently used tool to investigate the mechanical performance of structures under load. Here we apply its use to modeling the biomechanics of the zebrafish jaw.

Here, we present a protocol to demonstrate the generation of ice when water is introduced to a cold bath of brine, as a secondary refrigerant, at a range of temperatures well below the freezing point of water. It can be used as an alternative way of producing ice for industry.

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.

A protocol for stroke onset time estimation in a rat model of stroke exploiting quantitative magnetic resonance imaging (qMRI) parameters is described. The procedure exploits diffusion MRI for delineation of the acute stroke lesion and quantitative T₁ and T₂ (qT₁ and qT₂) relaxation times for timing of stroke.

Here we present a protocol to surgically create 'intestinal ligated loops' in chicken small intestines. This procedure allows for the comparison of multiple Clostridium perfringens strains' virulence in situ in a single host. This method markedly decreases the number of chickens usually necessary for similar in vivo experiments.

Repair of double-strand DNA breaks is a dynamic process, requiring not only formation of repair complexes at the breaks, but also their resolution after the lesion is addressed. Here, we use immunofluorescence microscopy for transient and long-lasting double-stranded breaks as a tool to dissect this genome maintenance mechanism.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

Here we present a protocol for live-imaging wound repair and the associated inflammatory response at high spatio-temporal resolution in vivo. This method utilizes the pupal stage of Drosophila development to enable long-term imaging and tracking of specific cell populations over time and is compatible with efficient RNAi-mediated gene inactivation.

We present a method that combines membrane protein purification and reconstitution into peptidiscs in a single chromatographic step. Biotinylated scaffolds are used for direct surface attachment and measurement of protein-ligand interactions via biolayer interferometry.

This protocol outlines the steps for inducing Primed Mycobacterial Uveitis (PMU) in mice. This method outlines the steps to help produce reliable and robust ocular inflammation in the mouse model system. Using this protocol, we generated uveitic eyes and uninflamed fellow eyes from single animals for further evaluation with immunologic, transcriptomic, and proteomic assays.

Here we present a protocol describing viral transduction of discrete brain regions with optogenetic constructs to permit synapse-specific electrophysiological characterization in acute rodent brain slices.

Breast cancer cells exhibit different dielectric properties compared to non-tumor breast epithelial cells. It has been hypothesized that, based on this difference in dielectric properties, the two populations can be separated for immunotherapy purposes. To support this, we model a microfluidic device to sort MCF-7 and MCF-10A cells.

APTrack is a software plugin developed for the Open Ephys platform that enables real-time data visualization and the closed-loop electrical threshold tracking of neuronal action potentials. We have successfully used this in microneurography for human C-fiber nociceptors and mouse C-fiber and Aδ-fiber nociceptors.

This protocol describes the harvesting and visualization of elasmoid scales of zebrafish during in vivo regeneration. In addition, the ex vivo culture of these scales for up to 7 days after harvest is presented.

Local anesthetic thoracoscopy (LAT) is essential for diagnosing recurrent, undiagnosed pleural effusion when a guideline-based workup fails to provide a specific cause. LAT can be performed as a day-case procedure by chest physicians. Here, we present a step-by-step approach for a successful and safe procedure.

Transthoracic ultrasound-guided lung biopsy represents a safe, cost-effective, and efficient approach for patients presenting with subpleural lung lesions suspected of malignancy. Employing a systematic, step-by-step process is crucial to achieve optimal patient selection, minimize complication risks, and maximize diagnostic accuracy.

Here, we present a feasibility study to assess a portable amplitude-integrated electroencephalogram (aEEG) recording system during the transport of infants with suspected hypoxic ischemic encephalopathy (HIE).