University of California, Davis

Non-fouling PEG silane monolayer was desorbed from individually addressable ITO electrodes on glass by application of a reductive potential. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.

We describe a method for preparing DNA coated gold bullets and demonstrate the use of such bullets to biolistically transfect neurons in cultured hippocampal slices.

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and α2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

We established mouse models of periventricular leukomalacia (PVL), the predominant brain injury in premature infants characterized by periventricular white matter lesions. Hypoxia/ischemia with/without systemic infection are the primary causes of PVL. Unilateral carotid ligation and hypoxia exposure with/without lipopolysaccharide injection creates PVL-like lesions in P6 mice.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

A combination of three single wavelength short-pulsed lasers is used to generate coherent anti-Stokes Raman scattering (CARS) and doubly-resonant CARS (DR-CARS). The difference between these signals provides enhanced sensitivity for otherwise difficult to detect coherent Raman signals, enabling imaging of weak Raman scatterers.

We describe procedures for recording daily locomotor activity rhythms of Drosophila and subsequent data analysis. Locomotor activity rhythms are a reliable behavioral output of animal circadian clocks and are used as the standard readout of clock function when studying circadian mutants or examining how the environment regulates the circadian system.

We provide a step-by-step protocol for whole-cell patch clamp recording of Calcium Release-Activated Calcium (CRAC) currents in peripheral blood mononuclear cell-derived human T lymphocytes.

Oligonucleotides can be used to site specifically substitute a single nucleotide of transfected target genes in both Anopheles gambiae and Anopheles stephensi cells.

The manufacture, calibration and use of non-invasive vibrating probes to measure bioelectric current in various biological systems is described.

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single sensillum recordings (SSR), which are summed responses from all odorant receptor neurons (ORNs) located on the antenna and from those housed in individual sensilla, respectively.

We demonstrate the use of DNA microarrays for expression profiling of the nervous system. We describe RNA quality control, sample labeling, and array hybridization and scanning.

We discuss the construction and operation of a complex nonlinear optical system that uses ultrafast all-optical switching to isolate Raman from fluorescence signals. Using this system we are able to successfully separate Raman and fluorescence signals utilizing pulse energies and average powers that remain biologically safe.

Lentiviruses are a valuable research tool for exploring gene function; however, researchers may wish to avoid production of pantropic lentivirus encoding known or suspected oncogenes. As an alternative, we present a safer protocol for use of ecotropic lentivirus on human cells modified to express the ecotropic receptor mSlc7a1.

This study describes diagnosis of avian influenza in wild birds using a portable rRT-PCR system. The method takes advantage of freeze-dried reagents to screen wild birds in a non-laboratory setting, typical of an outbreak scenario. Use of molecular tools provides accurate and sensitive alternatives for rapid diagnosis.

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

Magnetic resonance imaging (MRI) provides a powerful tool to evaluate the effectiveness of process equipment during operation. We discuss the use of MRI to visualize mixing in a static mixer. The application is relevant to personal care products, but can be applied to a broad range of food, chemical, biomass and biological fluids.

We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

A rapid method for volatile compound analysis in fruit is described. The volatile compounds present in the headspace of a homogenate of the sample are rapidly separated and detected with ultra-fast gas chromatography (GC) coupled with a surface acoustic wave (SAW) sensor. A procedure for data handling and analysis is also discussed.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

Surface renewal is a micrometeorological method that is being used increasingly to determine energy fluxes, but its technical complexity makes it inaccessible to a broad audience. We describe the steps needed to set up and calibrate a surface renewal field station, to acquire and process data, and to correctly interpret results.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

The robust device design of fringing-field electrostatic MEMS actuators results in inherently low squeeze-film damping conditions and long settling times when performing switching operations using conventional step biasing. Real-time switching time improvement with DC-dynamic waveforms reduces the settling time of fringing-field MEMS actuators when transitioning between up-to-down and down-to-up states.

A protocol involving integrated concentration, enrichment, and end-point colorimetric detection of foodborne pathogens in large volumes of agricultural water is presented here. Water is filtered through Modified Moore Swabs (MMS), enriched with selective or non-selective media, and detection is performed using paper-based analytical devices (µPAD) imbedded with bacterial-indicative colorimetric substrates.

The Focus Formation Assay provides a straightforward method to assess the transforming potential of a candidate oncogene.

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

We present a method to apply a physiological electric field to migrating, immortalized prostate cells in a custom-made galvanotaxis chamber. Using this method, we demonstrate that 2 lines of non-tumorigenic prostate cells demonstrate different degrees of migration directionality in the field.

Spinal cord injury models should be highly reproducible. We demonstrate that the calibrated forceps compression model of spinal cord injury is an easy to use surgical method for generating reproducible injuries to the murine spinal cord.

The durability of polymers and fiber-reinforced polymer composites in service is a critical aspect for their designs and condition-based maintenance. We present a novel low-cost laboratory testing platform for the investigation of the influence of concurrent mechanical and environmental loadings, and may help design more efficient yet safer composite structures.

The method presented here uses simultaneous positron emission tomography and magnetic resonance imaging. In the cerebral hypoxia-ischemia model, dynamic changes in diffusion and glucose metabolism occur during and after injury. The evolving and irreproducible damage in this model necessitates simultaneous acquisition if meaningful multi-modal imaging data are to be acquired.

Transporters in cell membranes allow differential segregation of ions across cell membranes or cell layers and play crucial roles during tissue physiology, repair and pathology. We describe the ion-selective self-referencing microelectrode that allows the measurement of specific ion fluxes at single cells and tissues in vivo.

Here, we outline how to study mitochondrial localization of a (cell cycle) kinase, and how to determine its sub-mitochondrial location as well as potential mitochondrial substrates/targets. Forced expression of proteins into the mitochondria provides a useful tool for studying the functional consequences of mitochondrial localization of a protein of interest.

In this study, a nano-microfluidic flow chamber was employed to visualize and functionally characterize the twitching motility of Xylella fastidiosa, a bacterium that causes Pierce's disease in grapevine.

This protocol shows how to perform cytoplasmic microinjection in farm animal zygotes. This technique can be used to deliver any solution into the one-cell embryo such as genome editing tools to generate knockout animals.

Here we present a protocol that facilitates the medium to high throughput functional characterization of gene and promoter constructs in tree secondary stem tissue within comparatively short time frames. It is efficient, easy to use and widely applicable to a range of tree species.

To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

A viable technique for the formation of strontium titanate bicrystals at high pressure and fast heating rate via the spark plasma sintering apparatus is developed.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

This article summarizes the design attributes and the effectiveness of treatment systems that treat urban stormwater and agriculture irrigation runoff to remove pesticides and other contaminants associated with aquatic toxicity.

Unlike ubiquitin ligases, few E3 SUMO ligases have been identified. This modified in vitro SUMOylation protocol is able to identify novel SUMO E3 ligases by an in vitro reconstitution assay.

Viral transcriptional factories are discrete structures that are enriched with cellular RNA polymerase II to increase viral gene transcription during reactivation. Here, a method to locate sites of actively transcribing viral chromatin in 3D nuclear space by a combination of immunofluorescence staining and in situ RNA hybridization is described.

We describe methods to isolate canine neutrophils from whole blood and visualize NET formation in live neutrophils using fluorescence microscopy. Also described are protocols to quantify NET formation and citrullinated histone H3 (citH3) expression using immunofluorescence microscopy.

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1^-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce apoptosis. We present an in vitro PDT protocol designed to simulate PDT that may be used to study differences in PS incubation and light treatment parameters.

Here, we present a protocol to transiently improve cardiac function in Duchenne muscular dystrophy mice by transplanting exosomes derived from normal myogenic progenitor cells.

Here, we present a Cas9-based exon23 deletion protocol to restore dystrophin expression in iPSC from Dmd^mdx mouse-derived skin fibroblasts and directly differentiate iPSCs into myogenic progenitor cells (MPC) using the Tet-on MyoD activation system.

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).

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Bioremediation is the capacity of organisms to degrade contaminants. Here, we describe methodologies to isolate and test microbes presenting bioremediation ability and potential probiotic characteristics for corals.

Nuclear surface spreads are an indispensable tool for studying chromosome events during meiosis. Here we demonstrate a method to prepare and visualize meiotic chromosomes during prophase I from zebrafish spermatocytes.

Described here is a proximity labeling method for identification of interaction partners of the TIR domain of the NLR immune receptor in Nicotiana benthamiana leaf tissue. Also provided is a detailed protocol for the identification of interactions between other proteins of interest using this technique in Nicotiana and other plant species.

We describe a method to identify neutrophil extracellular traps (NETs) in formaldehyde-fixed and paraffin-embedded feline cardiogenic arterial thrombi using heat-induced antigen retrieval and a double immunolabeling protocol.

Palmitoylation entails the incorporation of a 16-carbon palmitate moiety to cysteine residues of target proteins in a reversible manner. Here, we describe a biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest in mouse brain lysates.

This protocol describes a method to determine the influence of ryegrass residue addition on soil organic matter mineralization (i.e., priming effect) as well as explore the changes in soil microbial biomass size induced by soil organic matter priming, which involves artificially changing the size of microbial biomass.

The protocol has been developed to effectively extract intact histones from sorghum leaf materials for profiling of histone post-translational modifications that can serve as potential epigenetic markers to aid engineering drought resistant crops.

A protocol for synthesizing ~12 nm diameter gold nanoparticles (Au nanoparticles) in an organic solvent is presented. The gold nanoparticles are capped with oleylamine ligands to prevent agglomeration. The gold nanoparticles are soluble in organic solvents such as toluene.

Described here is a DNA extraction protocol using magnetic beads to produce high quality DNA extractions from mosquitoes. These extractions are suitable for a downstream next-generation sequencing approach.

This protocol describes a novel rAAV-based transient enhancer-reporter assay. This assay can be used to induce enhancer-driven expression in vivo in the mouse brain.

This protocol describes a robust method for using high-throughput settings to screen the antibacterial efficacy of bacteriophage cocktails.

This article describes encapsulation of human pluripotent stem cells (hPSCs) using a co-axial flow focusing device. We demonstrate that this microfluidic encapsulation technology enables efficient formation of hPSC spheroids.

This article focuses on the use of electronic absorption spectroscopy and isothermal titration calorimetry to probe and quantify the thermodynamics of Cu(II) binding to peptides and proteins.

Polyp bail-out is a process induced by acute stress, in which coral polyps digest the tissue connecting them to their colony and detach from it to live as individuals. The present protocol describes how to induce coral micropropagation by bail-out using hypersaline or calcium-free seawater treatments.

The protocol describes how to generate knock-out myoblasts using CRISPR/Cas9, starting from the design of guide-RNAs to the cellular cloning and characterization of the knock-out clones.

The D-loop capture (DLC) and D-loop extension (DLE) assays utilize the principle of proximity ligation together with quantitative PCR to quantify D-loop formation, D-loop extension, and product formation at the site of an inducible double-stranded break in Saccharomyces cerevisiae.

This protocol describes the procedure for genome editing in mouse bone marrow-derived macrophages using Cas9-sgRNA ribonucleoprotein complexes assembled in vitro and delivered by electroporation.