University of Iowa

Procedure for fertilizing Xenopus oocytes by the host transfer method.

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

A combination of different techniques to maximize data collection from mouse tissue is presented.

This video shows an effective technique for differentiating and dissecting the various semi-transparent structures of the human vitreous body in post mortem eyes.

The dissection technique illustrates evisceration of the vitreous, retina, and lens from the mouse eye, separation by centrifugation, and characterization with protein assays.

The dissection technique illustrates enucleation of the mouse eye for tissue fixation to perform phenotyping in high-throughput screens.

Time-lapse microscopy and image processing techniques were used to observe and analyze fibroblast-mediated gel compaction and fibrin fiber realignment in an environmentally controlled bioreactor over a 48 hr period.

Disease-causing mutations in actin can alter cytoskeletal function. Cytoskeletal dynamics are quantified through imaging of fluorescently tagged proteins using total internal fluorescence microscopy. As an example, the cytoskeletal protein, Aip1p, has altered localization and movement in cells expressing the mutant actin isoform, R256H.

Here we present a novel Ca²⁺-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca²⁺ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

The human retina is composed of functionally and molecularly distinct regions, including the fovea, macula, and peripheral retina. Here, we describe a method using punch biopsies and manual removal of tissue layers from a human eye to dissect and collect these distinct retinal regions for downstream proteomic analysis.

Batch processing of yeast 2-hybrid screens allows for direct comparison of the interaction profiles of multiple bait proteins with a highly complex set of prey fusion proteins. Here, we describe refined methods, new reagents, and how to implement their use for such screens.

Deep sequencing of yeast populations selected for positive yeast 2-hybrid interactions potentially yields a wealth of information about interacting partner proteins. Here, we describe the operation of specific bioinformatics tools and customized updated software to analyze sequence data from such screens.

Here, we describe two measures of pulmonary function – barometric plethysmography, which allows the measurement of lung volume, and volumetric capnography, a tool to measure the anatomic dead space and airways uniformity. These techniques may be used independently or combined to assess airways function at different lung volumes.

A protocol to create gene modified human pseudoislets from dispersed human islet cells that are transduced by lentivirus carrying short hairpin RNA (shRNA) is presented. This protocol utilizes readily available enzyme and culture vessels, can be performed easily, and produces genetically modified human pseudoislets suitable for functional and morphological studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

Bacterial pathogens secrete proteins into the host that target crucial biological processes. Identifying the host pathways targeted by bacterial effector proteins is key to addressing molecular pathogenesis. Here, a method using a modified yeast suppressor and toxicity screen to elucidate host pathways targeted by toxic bacterial effector proteins is described.

Bench-scale, axenic cultivation facilitates microalgal characterization and productivity optimization before subsequent process scale-up. Photobioreactors provide the necessary control for reliable and reproducible microalgal experiments and can be adapted to safely cultivate microalgae with the corrosive gases (CO₂, SO₂, NO₂) from municipal or industrial combustion emissions.

The goal of this protocol is to describe the use of esophageal temperature modulation to counteract esophageal thermal injury from left atrial ablation for the treatment of atrial fibrillation.

This live-bacterial cell imaging protocol allows for visualization of interactions between multiple bacterial species at the single-cell level over time. Time-lapse imaging allows for observation of each bacterial species in monoculture or coculture to interrogate interspecies interactions in multispecies bacterial communities, including individual cell motility and viability.

We developed a model of chorioamnionitis to simulate fetal exposure to maternal inflammation (FEMI) without complications of live organisms to examine the effects of FEMI on development of the offspring’s intestinal tract. This allows for study of mechanistic causes for development of intestinal injury following chorioamnionitis.

This protocol describes how to generate bilateral, full-thickness excisional wounds in mice and how to subsequently monitor, harvest, and prepare the wounds for morphometric analysis. Included is an in-depth description of how to use serial histological sections to define, precisely quantify and detect morphometric defects.

Here we demonstrate a method to apply fluid shear stress to cancer cells in suspension to model the effects of hemodynamic stress on circulating tumor cells.

A standard protocol is described to study the antitumor activity and associated toxicity of IL-1α in a syngeneic mouse model of HNSCC.

Rodents are not able to report migraine symptoms. Here, we describe a manageable test paradigm (light/dark and open field assays) to measure light aversion, one of the most common and bothersome symptoms in patients with migraines.

Presented here is a protocol to perform comprehensive neonatal echocardiography by trained neonatologists in the neonatal intensive care unit. The trained individuals provide longitudinal assessments of heart function, systemic and pulmonary hemodynamics in a consultative role. The manuscript also describes the requirements to become a fully trained neonatal hemodynamics specialist.

This manuscript describes a method for screening moderately sized Candida albicans mutant libraries for morphogenesis phenotypes during active infection in a mammalian host using non-invasive confocal microscopy.

The present protocol explains the generation of a 2D monolayer of cerebellar cells from induced pluripotent stem cells for investigating the early stages of cerebellar development.

Here we describe a time-specific method to effectively manipulate critical developmental pathways in the mouse placenta in vivo. This is performed through the injection and electroporation of CRISPR plasmids into the placentas of pregnant dams on embryonic day 12.5.

This protocol presents an integrated biorepository platform for the standardized collection, annotation, and biobanking of high-quality human aqueous humor and vitreous liquid biopsies for molecular downstream analyses, including proteomics, metabolomics, and glycomics.

Patient-derived tumor organoids are a sophisticated model system for basic and translational research. This methods article details the use of multiplexed fluorescent live-cell imaging for simultaneous kinetic assessment of different organoid phenotypes.

This protocol provides a method for tracking automated eye squint in rodents over time in a manner compatible with time-locking to neurophysiological measures. This protocol is expected to be useful to researchers studying mechanisms of pain disorders such as migraine.