University of Michigan, Ann Arbor

Here we describe a data collection and data analysis method for functional Near Infrared Spectroscopy (fNIRS), a novel non-invasive brain imaging system used in cognitive neuroscience, particularly in studying child brain development. This method provides a universal standard of data acquisition and analysis vital to data interpretation and scientific discovery.

Here we describe a method to quantify infectious particles of murine norovirus (MNV), which is the only norovirus that efficiently replicates in cell culture. The plaque assay takes advantage of MNV’s tropism for murine macrophages and can be adapted for use with biological or environmental samples containing MNV.

Recent development in gene targeting tools makes production of knockout (KO) rabbits possible. In the present work, we generated five Apolipoprotein (Apo) C-III KO rabbits using Zinc Finger Nucleases (ZFN). This work demonstrated that ZFN is a highly efficient method to produce KO rabbits.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

An Achilles tenotomy and burn injury model of heterotopic ossification allows for the reliable study of trauma induced ectopic bone formation without the application of exogenous factors.

Chronic Thromboembolic Pulmonary Hypertension (CTEPH) and Right Ventricular (RV) dysfunction were induced in piglets by progressive obstruction of the pulmonary arteries. Consequences were remarkably similar to those observed in CTEPH patients. This animal model would be a very useful tool for pathophysiology and therapeutic experiments on CTEPH and RV failure.

We describe the detailed procedures and strategies to measure the mechanical properties and mechanical unfolding pathways of single protein molecules using an atomic force microscope. We also show representative results as a reference for selection and justification of good single protein molecule recordings.

Here, we present a protocol to produce a bispecific antibody GPC3-S-Fab in Escherichia coli. The purified GPC3-S-Fab has potent cytotoxicity against GPC3 positive liver cancer cells.

Substrates with stiffness in the kilopascal-range are useful to study the response of cells to physiologically relevant micro-environment stiffness. Using just a widefield fluorescence microscope, the Young's modulus of soft silicone gels can be determined using an indentation with a suitable sphere.

We present a method for the generation of in vitro self-sustained mitotic oscillations at the single-cell level by encapsulating egg extracts of Xenopus laevis in water-in-oil microemulsions.

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

Presented here is a protocol for efficient CRISPR/Cas9 ribonucleoprotein-mediated gene editing in mammalian cells using tube electroporation.

The following manuscript describes a novel method for developing a biologic, closed loop neural feedback system termed the composite regenerative peripheral nerve interface (C-RPNI). This construct has the ability to integrate with peripheral nerves to amplify efferent motor signals while simultaneously providing afferent sensory feedback.

Engineered tissues heavily rely on proper vascular networks to provide vital nutrients and gases and remove metabolic waste. In this work, a stepwise seeding protocol of endothelial cells and support cells creates highly organized vascular networks in a high-throughput platform for studying developing vessel behavior in a controlled 3D environment.

This protocol details a simple method that quantifies R-loop, a three-stranded nucleic acid structure that comprises of an RNA-DNA hybrid and a displaced DNA strand.

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

Systematic scoring of intestinal inflammation using a free computer-assisted system is a powerful tool to quantitatively compare histopathological changes in colitis models characterized by the presence of ulcers and inflammatory changes. Histological colitis score evaluation strengthens clinical observations and facilitates data interpretation.

The method presented here summarizes optimized protocols for assessing cellular bioenergetics in non-adherent mouse hematopoietic stem and primitive progenitor cells (HSPCs) using the extracellular flux analyzer to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of HSPCs in real time.

Here, we describe fabrication methodology for customizable carbon fiber electrode arrays for recording in vivo in nerve and brain.

This manuscript provides an innovative method for developing a biologic peripheral nerve interface termed the Muscle Cuff Regenerative Peripheral Nerve Interface (MC-RPNI). This surgical construct can amplify its associated peripheral nerve's motor efferent signals to facilitate accurate detection of motor intent and the potential control of exoskeleton devices.

This article introduces a simple method for expeditious production of giant unilamellar vesicles with encapsulated cytoskeletal proteins. The method proves to be useful for bottom-up reconstitution of cytoskeletal structures in confinement and cytoskeleton-membrane interactions.

The present protocol describes a standardized surgical method for the elastase-induced AAA model through the direct application of elastase to the adventitia of infrarenal abdominal aorta in mice.

A set of protocols are presented that describe the measurement of contractile function via sarcomere length detection along with calcium (Ca²⁺) transient measurement in isolated rat myocytes. The application of this approach for studies in animal models of heart failure is also included.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer an alternative to using animals for preclinical cardiotoxicity screening. A limitation to the widespread adoption of hiPSC-CMs in preclinical toxicity screening is the immature, fetal-like phenotype of the cells. Presented here are protocols for robust and rapid maturation of hiPSC-CMs.

Presented here is a protocol to reliably quantify the right and left ventricular function of donor hearts after cold preservation using an ex vivo perfusion system.

Here, we present a protocol to allow providers to perform focused cardiac ultrasound (FoCUS) in the clinical environment. We describe methods of transducer manipulation, review common pitfalls of transducer movements, and suggest tips to optimize phased array transducer use.

In this work, a robust method for the quantification of mating efficiency in the yeast Saccharomyces cerevisiae is described. This method is particularly useful for the quantification of pre-zygotic barriers in speciation studies.

Advancing the Assessment of Cardiac Injury and Function

This article presents a method for estimating same-day P300 speller Brain-Computer Interface (BCI) accuracy using a small testing dataset.

This protocol describes a lipofuscin accumulation model in highly differentiated and polarized human retinal pigment epithelial (RPE) cultures and an improved outer segment (OS) phagocytosis assay to detect the total OS consumption/degradation capacity of the RPE. These methods overcome the limitations of previous lipofuscin models and classical pulse-chase outer segment phagocytosis assays.

We have developed a cross-species comparative oncogenomics approach utilizing genomic analyses and functional genomic screens to identify and compare therapeutic targets in tumors arising in genetically engineered mouse models and the corresponding human tumor type.

We present CorrelationCalculator and Filigree, two tools for data-driven network construction and analysis of metabolomics data. CorrelationCalculator supports building a single interaction network of metabolites based on expression data, while Filigree allows building a differential network, followed by network clustering and enrichment analysis.

A procedure to extract cell-free DNA from vitreous and aqueous humor to perform molecular studies for diagnosing vitreoretinal lymphoma is established here. The method offers the ability to concurrently extract DNA from the cellular component of the sample or to reserve it for ancillary testing.

The food source for Caenorhabditis elegans in the lab is live Escherichia coli. Since bacteria are metabolically active, they present a confounding variable in metabolic and drug studies in C. elegans. A detailed protocol to metabolically inactivate bacteria using paraformaldehyde is described here.

This protocol outlines two methods for the quantitative analysis of mitophagy in pancreatic β-cells: first, a combination of cell-permeable mitochondria-specific dyes, and second, a genetically encoded mitophagy reporter. These two techniques are complementary and can be deployed based on specific needs, allowing for flexibility and precision in quantitatively addressing mitochondrial quality control.

Luteinizing hormone (LH) pulsatility is a hallmark of reproductive function. We describe a protocol for remote activation of specific neuronal populations linked to serial automated blood collection. This technique allows timed hormonal modulation, multiplexing, and minimizing manipulation effects on LH levels in conscious freely moving, and undisturbed animals.

This protocol describes the inverted emulsion method used to encapsulate a cell-free expression (CFE) system within a giant unilamellar vesicle (GUV) for the investigation of the synthesis and incorporation of a model membrane protein into the lipid bilayer.

Our purpose was to provide an updated, easy-to-follow guide on the fabrication and testing of epimysial electromyography electrodes. To that end, we provide instructions for material sourcing and a detailed walkthrough of the fabrication and testing process.

We introduce a novel device for measuring oxygen consumption rates (OCR) in retinal pigment epithelial (RPE) cultures. The device can measure OCR for weeks at a time on RPE grown on standard cell culture plates with standard media while the plates are in a standard cell culture incubator.