University of Sydney

An inexpensive, high throughput method for simultaneous detection of up to 43 molecular targets is described. Applications of mPCR/RLB include microbial typing and detection of multiple pathogens from clinical samples.

We described a procedure for the disaggregation of colorectal cancer (CRC) to produce viable single cells, which are then captured on customized antibody microarrays recognizing surface antigens (DotScan CRC microarray). Sub-populations of cells bound to the microarray can be profiled by fluorescence multiplexing using monoclonal antibodies tagged with fluorescent dyes.

Analysis of vestibular hair cell function is complicated by their location deep within the hardest part of the skull, the petrous temporal bone. Most functional hair cell studies have used acutely isolated hair cells. Here we describe a semi-intact preparation of mouse vestibular epithelium for electrophysiological and two-photon microscopy studies.

Individuals with intermittent claudication exhibit poor balance compared to healthy controls. Computerized dynamic posturography is an objective method for measuring an individual's postural responses to balance disturbances. This provides an objective reflection of the person’s ability to respond to situations under which sensory stimuli are altered and unexpected perturbations occur.

A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently tagged proteins simultaneously using the method of transient dominant selection is described here.

Ascorbate plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. Here we describe a medium-throughput, specific and inexpensive microplate assay for the determination of both intra- and extracellular ascorbate in cell culture.

A technique is demonstrated for the microsurgical procedure for heterotopic transplantation of hearts in mice, including simplified methods for donor harvesting and recipient vessel anastomosis.

Motor control and balance performance are known to deteriorate with age. This paper presents a number of standard noninvasive behavioral tests with the addition of a simple rotary stimulus to challenge the vestibular system and show changes in balance performance in a murine model of aging.

Colloidal probe nanoscopy can be used within a variety of fields to gain insight into the physical stability and coagulation kinetics of colloidal systems and aid in drug discovery and formulation sciences using biological systems. The method described within provides a quantitative and qualitative means to study such systems.

Remote ischemic preconditioning (RIP) is a method of conditioning tissues against damaging stress. We have established a method of remote ischemia at the hind limb, by inflating a sphygmomanometer cuff for 5-10 min. The neuroprotective capabilities of RIP have been demonstrated in a model of retinal degeneration in rodents.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

Environmental enrichment provides a potential protective effect against neurodegenerative disorders. Currently, however, there is no easy way of determining the efficacy of enrichment procedures. This protocol describes a simple “Puzzle Box” method for assessing an animal’s cognitive function, in order to reveal the effectiveness of environmental enrichment.

Mitochondrial dysfunction is a hallmark of cellular senescence. This paper uses non-invasive near-infrared (NIr) treatment to improve mitochondrial function in the aging mouse vestibular sensory epithelium.

We describe the design and construction of an electrochemical cell for the examination of electrode materials using in situ neutron powder diffraction (NPD). We briefly comment on alternate in situ NPD cell designs and discuss methods for the analysis of the corresponding in situ NPD data produced using this cell.

The electroretinogram (ERG) is an electrical potential generated by the retina in response to light. This paper describes how to use the ERG to assess retinal function, in dark-adapted rats, and how it can be can be used to assess a neuroprotective intervention, in the present case remote ischemic preconditioning.

The fenestrated liver sinusoidal endothelial cell is a biologically important filter system that is highly influenced by various diseases, toxins, and physiological states. These changes significantly impact on liver function. We describe methods for the standardisation of the measurement of the size and number of fenestrations in these cells.

We describe a technique for inscribing identical fiber Bragg gratings into each core of a multicore fiber. This is achieved by introducing an additional surface into the optical path to mitigate lensing by the curved surface of the fiber cladding.

We describe two complementary methods using the fluorescence ubiquitination cell cycle indicator (FUCCI) and image analysis or flow cytometry to identify and isolate cells in the inner G1 arrested and outer proliferating regions of 3D spheroids.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

Detailed and generalized protocols are presented for the synthesis and subsequent purification of four palladium N-heterocyclic carbene complexes from benzimidazolium salts. The complexes were tested for catalytic activity in arylation and Suzuki-Miyaura reactions. For each reaction investigated, at least one of the four complexes successfully catalyzed the reaction.

The canine brain is a valuable model in which to study adult neurogenesis. Presented here are protocols for isolating and expanding adult canine hippocampal neural precursor cells from primary brain tissue.

This protocol describes the induction of an ischemia-reperfusion (IR) model on mouse ear skin using magnet clamping. Using a custom-built intravital imaging model, we study in vivo inflammatory responses post-reperfusion. The rationale behind the development of this technique is to extend the understanding of how leukocytes respond to skin IR injury.

This protocol describes the production of a therapeutic antibody in a mammalian expression system. The methods described include preparation of vector DNA, stable transfection and serum-free adaptation of a human embryonic kidney 293 cell line, set up of large scale cultures and purification using affinity chromatography.

This study describes a protocol that uses ¹⁸F-FDG and positron emission tomography/computed tomography (PET/CT) imaging, together with kinetic modelling, to quantify the in vivo, real-time uptake of ¹⁸F-FDG into tissues.

This protocol demonstrates the induction of cerebral cavernous malformation disease in a mouse model and uses contrast enhanced micro computed tomography to measure lesion burden. This method enhances the value of established mouse models to study the molecular basis and potential therapies for cerebral cavernous malformation and other cerebrovascular diseases.

This study implemented whole genome sequencing for analysis of mutations in genes conferring antifungal drug resistance in Candida glabrata. C. glabrata isolates resistant to echinocandins, azoles and 5-flucytosine, were sequenced to illustrate the methodology. Susceptibility profiles of the isolates correlated with presence or absence of specific mutation patterns in genes.

This protocol describes a reproducible and reliable method for the sublimation-based preparation of formalin fixed tissue destined for imaging mass spectrometry.

The 'Boden Food Plate' is an electronic food diary designed to be an interactive and fun method to collect dietary intake using visual depictions. The purpose of this study was to validate the web-based application against a traditional three-day estimated food diary method.

Here, we present a protocol to establish a replicable conductive hearing loss induction via surgical tympanic membrane puncture and verification by otoscope visualization and behavioral assessment by clap startle.

Full-length individual proviral sequencing (FLIPS) provides an efficient and high-throughput method for the amplification and sequencing of single, near full-length (intact and defective) HIV-1 proviruses and allows for determination of their potential replication-competency. FLIPS overcomes limitations of previous assays designed to sequence the latent HIV-1 reservoir.

Providing single-cell sensitivity, real-time flow cytometry is uniquely suited to quantify multimodal receptor functions of live cultures. Using adult neural progenitor cells, the P2X7 receptor function was assessed via calcium influx detected by calcium indicator dye, transmembrane pore formation by ethidium bromide uptake, and phagocytosis using fluorescent latex beads.

Here, we present detailed instructions on how to build and calibrate research quality ceptometers (light sensors that integrate light intensity across many sensors arrayed linearly along a horizontal bar).

Protocols are presented to assess the autonomic and behavioral effects of passive motion in rodents using elevator vertical motion and Ferris-wheel rotation.

Galvanic vestibular stimulation in humans exhibits improvements in vestibular function. However, it is unknown how these effects occur. Here, we describe how to apply sinusoidal and stochastic electrical noise and evaluate appropriate stimulus amplitudes in individual medial vestibular nucleus neurons in the C57BL/6 mouse.

Presented here is a protocol to detect macrophage extracellular trap (MET) production in live cell culture using microscopy and fluorescence staining. This protocol can be further extended to examine specific MET protein markers by immunofluorescence staining.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

In the clinical context, patients with localized pancreatic cancer will undergo pancreatectomy followed by adjuvant treatment. This protocol reported here aims to establish a safe and effective method of modelling this clinical scenario in nude mice, through orthotopic implantation of pancreatic cancer followed by distal pancreatectomy and splenectomy.

This protocol aims to fabricate 3D cardiac spheroids (CSs) by co-culturing cells in hanging drops. Collagen-embedded CSs are treated with doxorubicin (DOX, a cardiotoxic agent) at physiological concentrations to model heart failure. In vitro testing using DOX-treated CSs may be used to identify novel therapies for heart failure patients.

A portable system capable of measuring steady-state visual-evoked potentials was developed and trialed on 65 amateur rugby players over 18 weeks to investigate SSVEP as a potential electrophysiological biomarker for concussion. Players' baselines were measured pre-season, with retesting for reliability, concussion, and recovery assessment being conducted within controlled time-periods, respectively.

We present a suite of standardized single- and paired-pulse transcranial magnetic stimulation (TMS) recording protocols, with options for conventional amplitude measurements and threshold-tracking. This program can control three different types of magnetic stimulators and is designed to enable all tests to be performed conveniently by a single operator.

This protocol describes a 3D printing technique to fabricate bone-like structures by depositing a calcium phosphate ink in a gelatin-based granular support. Printed bone analogs are deposited in freeform, with flexibility for direct harvesting of the print or crosslinking within a living cell matrix for multiphasic constructs.

Cardiothoracic surgical models in mice >7 days old require intubation, but this is challenging for preadolescent (8-14-day-old) mouse pups and there is little information on anesthetic regimens for intubation. Here, we present dosage regimens of ketamine/xylazine/atropine in 10-day-old C57BL/6J mouse pups that allow endotracheal intubation, while minimizing animal mortality.

Tumor spheroids are becoming increasingly utilized to assess tumor cell-microenvironment interactions and therapy response. The present protocol describes a robust but simple method for semi-high-throughput imaging of 3D tumor spheroids using rapid optical clearing.

Skeletal muscle function can be assessed by quantifying the contractility of isolated muscle fibers, traditionally using laborious, low-throughput approaches. Here, we describe an optics-based, high-throughput method to quantify the contractility of hydrogel-embedded muscle fibers. This approach has applications for drug screening and therapeutic development.

The present article describes the methodological considerations for the noninvasive assessment of abdominal aortic and carotid intima-media thickness using B-mode ultrasonography. This technique is commonly used in the developmental origins of health and disease research as a surrogate for early arterial changes.