University of California, Los Angeles

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy. Insight is given into how computational models can provide information on the population dynamics of mosquitoes and the spread of transposable elements through A. gambiae subspecies. The ethical considerations of releasing genetically modified mosquitoes into the wild are discussed.

Cortical blood flow dynamics can be studied in vivo by imaging fluorescent dextran dyes injected into the tail vein of rodents with 2-photon microscopy. This video shows how to image blood flow dynamics in neocortex of mice through a glass-covered cranial window preparation.

This video and protocol demonstrate how to implant a glass-covered cranial window in rodents. These preparations can be used for chronic in vivo two-photon imaging of the neocortex over time scales of months. It may also be used for other types of imaging, including optical intrinsic signal imaging.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells. Part 1 of 3.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 2 of 3.

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 3 of 3.

Here we describe how to tether a fly in an olfactory magnetic-tether (OMT) apparatus. We describe how to align the rare-earth magnets and odor ports, and how to set mass flow rates for both the stimulus delivery and vacuum suction to achieve optimal odor tracking.

Photo-induced cross-linking of unmodified proteins (PICUP) allows characterization of oligomer size distribution in metastable protein mixtures. We demonstrate application of PICUP to three representative amyloidogenic peptides the 40- and 42-residue forms of amyloid β-protein, and calcitonin, and a control peptide growth-hormone releasing factor.

Here we describe how to optimize the acquired video image for an olfactory magnetic-tether (OMT) apparatus. We also describe two sample experimental protocols for studying visuo-olfactory fusion.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

Lensfree on-chip imaging and characterization of cells is illustrated. This on-chip cell imaging approach provides a compact and cost-effective tool for medical diagnostics and high-throughput cell biology applications, making it especially suitable for resource poor settings.

The Lashley III maze is a route-learning task that does not rely on aversive stimuli or visual cues. It is thus a highly attractive option for evaluating learning and memory, especially in aging mice or otherwise where stress is a consideration.

Aptamers are short ribo-/deoxyribo-oligonucleotides selected by in-vitro evolution methods based on affinity for a specific target. Aptamers are molecular recognition tools with versatile therapeutic, diagnostic, and research applications. We demonstrate methods for selection of aptamers for amyloid β-protein, the causative agent of Alzheimer's disease.

mStrawberry OP9 cells allow for complete evaluation of all ES-derived progeny from co-culture.

An efficient method to assess surface-exposure of leptospiral proteins is described. The method is specifically designed to avoid disruption of the fragile outer membrane of leptospiral cells. This technique requires employment of several negative controls to assess the integrity of the outer membrane and specificity of antibody reaction.

Fopius arisanus is an egg-larval parasitoid of Tephritid fruit flies that is successfully used in biological control of these important tropical pests. We describe here an optimized protocol for rearing F. arisanus on a small scale using readily available materials.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

A reliable method to investigate outer hair cell (OHC) motile responses, including electromotility, slow motility and bending, is described. OHC motility is elicited by stimulation with an external alternating electrical field, and the method takes advantage of high-speed image recording, LED-based illumination, and last generation image analysis software.

Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.

A lensless on-chip fluorescent microscopy platform is demonstrated that can image fluorescent objects over an ultra-wide field-of-view of e.g., >0.6-8 cm2 with <4μm resolution using a compressive sampling based decoding algorithm. Such a compact and wide-field fluorescent on-chip imaging modality could be valuable for high-throughput cytometry, rare-cell research and microarray-analysis.

We present a protocol for concurrent collection of EEG/fMRI data, and synchronized MR clock signal recording. We demonstrate this method using a unique paradigm whereby subjects receive ‘cold glove’ instructions during scanning, and EEG/fMRI data are recorded along with hand temperature measurements both before and after hypnotic induction.

Lensfree optical tomography is a three-dimensional microscopy technique that offers a spatial resolution of <1 μm × <1 μm × <3 μm in x, y and z dimensions, respectively, over a large imaging-volume of 15-100 mm³, which can be particularly useful for integration with lab-on-a-chip platforms.

By combining a polished and reinforced thin-skull (PoRTS) cranial window and glioblastoma (GBM) cell injection, we can observe glioma initiation and growth from injected GBM cells in the brain of a live mouse longitudinally.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

Optogenetic techniques have made it possible to study the contribution of specific neurons to behavior. We describe a method in larval zebrafish for activating single somatosensory neurons expressing a channelrhodopsin variant (ChEF) with a diode-pumped solid state (DPSS) laser and recording the elicited behaviors with a high-speed video camera.

In this video, we will demonstrate how to record electrical activity from identified single neurons in a whole brain preparation, which preserves complex neural circuits. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with a fluorescent protein for identification in the intact brain preparation.

This article details the procedures for optical imaging analysis of the tumor-targeted nanoparticle, HerDox. In particular, detailed use of the multimode imaging device for detecting tumor targeting and assessing tumor penetration is described here.

We review our recent results on the integration of fluorescent microscopy and imaging flow cytometry tools on a cell-phone using compact and cost-effective opto-fluidic attachments. These cell-phone based micro-analysis devices might be useful for cytometric analysis, such as performing various cell counting tasks as well as for high-throughput screening of e.g., water samples in resource limited settings.

Parabiotic joining of two organisms leads to the development of a shared circulatory system. In this protocol, we describe the surgical steps to form a parabiotic connection between a wild-type mouse and a constitutive GFP-expressing mouse.

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

The Default Mode Network (DMN) in Temporal Lobe Epilepsy (TLE) is analyzed in the resting state of the brain using seed-based functional connectivity MRI (fcMRI).

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

Exosomes are microvesicular structures found within biofluids that potentially carry important disease discriminatory biomarkers. Here, a novel method is used to specifically extract exosomes and rapidly test the exosomal cargo for both RNA/protein targets following the disruption of exosomes using non-uniform electric cyclic square waves.

We describe the detailed steps of a high-throughput chemical assay in the nematode Caenorhabditis elegans used to assess germline toxicity. In this assay, disruption of germline function following chemical exposure is monitored using a fluorescent reporter specific to aneuploid embryos.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

An efficient, three-step synthesis of RAFT-based fluorescent glycopolymers, consisting of glycomonomer preparation, copolymerization, and post-modification, is demonstrated. This protocol can be used to prepare RAFT-based statistical glycopolymers with desired structures.

Attention control comprises enhancement of target signals and attenuation of distractor signals. We describe an approach to measure separately but concurrently, the neurophysiology of attending and ignoring in sustained intermodal attention, utilizing a passive control condition during which neither process is continuously engaged.

Here we present methodology for the production of a focal stroke in murine white matter by local injection of an irreversible endothelial nitric oxide synthase (eNOS) inhibitor (L-Nio). Presented are two stereotactic variations, retrograde neuronal tracing, and fresh tissue labeling and dissection that expand the potential applications of this technique.

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection.

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs.

House Ear Institute-Organ of Corti 1 (HEI-OC1) is one of the few mouse auditory cell lines currently available for research purposes. This protocol describes how to work with HEI-OC1 cells to investigate the cytotoxic effects of pharmacological drugs as well as functional properties of inner ear proteins.

Understanding the cellular and molecular mechanisms of re-endothelialization following arterial denudation injury is of paramount importance in preventing thrombosis and restenosis of arteries. Here we describe a protocol for reproducible arterial denudation injury of the infrarenal abdominal aorta. The procedure was developed to investigate the underlying mechanisms that regulate endothelial regeneration using mouse models.

Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and discussed.

Subretinal injections are the most common technique for delivering large therapeutic agents such as proteins and viral vectors to photoreceptors and the retinal pigment epithelium. An alternative method in mice that successfully targets the subretinal space with minimal collateral damage and fast recovery times is described here.

We describe a method for generating localization and affinity purification (LAP)-tagged inducible stable cell lines for investigating protein function, spatiotemporal subcellular localization and protein-protein interaction networks.

This manuscript describes how to implement a psychophysiological interaction analysis to reveal task-dependent changes in functional connectivity between a selected seed region and voxels in other regions of the brain. Psychophysiological interaction analysis is a popular method to examine task effects on brain connectivity, distinct from traditional univariate activation effects.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

We describe a protocol to measure transmigration by monocytes across human endothelial monolayers and their subsequent maturation into foam cells. This provides a versatile method to assess the atherogenic properties of monocytes isolated from people with different disease conditions and to evaluate factors in blood which may enhance this propensity.

We describe here a fluorometric cell-free biochemical assay for determination of HDL-lipid peroxidation. This rapid and reproducible assay can be used to determine HDL function in large scale studies and can contribute to our understanding of HDL function in human disease.

We describe a protocol for generating proliferating and quiescent primary human dermal fibroblasts, monitoring transcript decay rates, and identifying differentially decaying genes.

High-frequency ultrasound imaging of the fetal mouse has improved imaging resolution and can provide precise non-invasive characterization of cardiac development and structural defects. The protocol outlined herein is designed to perform real-time fetal mice echocardiography in vivo.

Mice individually housed with a running wheel while given limited access to food develop reductions in food consumption and increase activity on the running wheel. This experimental phenomenon is called activity-based anorexia. This paradigm provides an experimental tool for studying the neurobiology and behaviors underlying aspects of anorexia nervosa.

We describe a protocol for mapping the spatial distribution of enzymatic activity for enzymes that generate nicotinatmide adenine dinucleotide phosphate (NAD(P)H) + H+ directly in tissue samples.

Here we use bioluminescent, X-ray, and positron-emission tomography/computed tomography imaging to study how inhibiting mTOR activity impacts bone marrow-engrafted myeloma tumors in a xenograft model. This allows for physiologically relevant, non-invasive, and multimodal analyses of the anti-myeloma effect of therapies targeting bone marrow-engrafted myeloma tumors in vivo.

Here, we present a protocol for three-dimensional culture of patient-derived glioblastoma cells within orthogonally tunable biomaterials designed to mimic the brain matrix. This approach provides an in vitro, experimental platform that maintains many characteristics of in vivo glioblastoma cells typically lost in culture.

Threats reliably evoke shifts in high-level ideological investment, but little work to date has explored the neural mechanisms underlying these dynamics. This paper describes how continuous theta burst transcranial magnetic stimulation may be employed to test the contribution of the posterior medial frontal cortex (and/or other regions) to threat-related ideological shifts.

Here we describe the detailed methodology required to conduct stress-enhanced fear learning (SEFL) experiments, a preclinical model of post-traumatic stress disorder, in both rats and mice. The model utilizes aspects of Pavlovian fear conditioning and freezing as an index of enhanced fear in rodents.

We present a protocol and associated metadata template for the extraction of text describing biomedical concepts in clinical case reports. The structured text values produced through this protocol can support deep analysis of thousands of clinical narratives.

Here we present simplified methods for fabricating transparent neurovascular phantoms and characterizing the flow therein. We highlight several important parameters and demonstrate their relationship to field accuracy.

We present a protocol and associated programming code as well as metadata samples to support a cloud-based automated identification of phrases-category association representing unique concepts in user selected knowledge domain in biomedical literature. The phrase-category association quantified by this protocol can facilitate in depth analysis in the selected knowledge domain.

Traditional cDNA-based overexpression techniques have a limited applicability for the overexpression of long noncoding RNAs due to their multiple splice forms with potential functionality. This review reports a protocol using CRISPR technology to overexpress multiple splice variants of a long noncoding RNA.

The purpose of this manuscript and protocol is to explain and demonstrate in detail the surgical procedure of orthotopic kidney transplantation in rats. This method is simplified to achieve the correct perfusion of the donor kidney and shorten the reperfusion time by using the venous and ureteral cuff anastomosis technique.

This protocol demonstrates how to prepare a briquette sample and conduct a uniaxial compression experiment with a briquette in different CO₂ pressures using a visualized and constant-volume gas-solid coupling test system. It also aims to investigate changes in terms of coal’s physical and mechanical properties induced by CO₂ adsorption.

Here, we present a reliable, minimally invasive, and cost-effective method to record and interpret electrocardiograms in live anesthetized adult zebrafish.

Immunoglobulin G (IgG) N-glycan is characterized using hydrophilic interaction chromatography UPLC. In addition, the structure of IgG N-glycan is clearly separated. Presented here is an introduction to this experimental method so that it can be widely used in research settings.

Mouse prostate organoids represent a promising context to evaluate mechanisms that regulate differentiation. This paper describes an improved approach to establish prostate organoids, and introduces methods to (1) collect protein lysate from organoids, and (2) fix and stain organoids for whole-mount confocal microscopy.

Metastatic clear cell renal cell carcinoma is a disease without a comprehensive animal model for thorough preclinical investigation. This protocol illustrates two novel animal models for the disease: the orthotopically implanted mouse model and the chicken chorioallantoic membrane model, both of which demonstrate lung metastasis resembling clinical cases.

Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC.

Curcumin is an ideal fluorophore for labeling and imaging of amyloid beta protein plaques in brain tissue due to its preferential binding to amyloid beta protein as well as its structural similarities with other traditional amyloid binding dyes. It can be used to label and image amyloid beta protein plaques more efficiently and inexpensively than traditional methods.

Stroke is a global issue with minimal treatment options and no current clinical therapy for regenerating the lost brain tissue. Here we describe methods for creating precise photothrombotic stroke in the motor cortex of rodents and subsequent injection of hydrogel biomaterials to study their effects on tissue regeneration after stroke.

Presented here is a protocol for chronic sleep fragmentation (CSF) model achieved by an electrically controlled orbital rotor, which could induce confirmed cognitive deficit and anxiety-like behavior in young wild-type mice. This model can be applied to explore the pathogenesis of chronic sleep disturbance and related disorders.

A protocol to co-inject cancer cells and fibroblasts and monitor tumor growth over time is provided. This protocol can be used to understand the molecular basis for the role of fibroblasts as regulators of tumor growth.

In this manuscript, we discuss a novel method to sample and analyze the duodenal microbiome. This method provides an accurate depiction of microbial diversity and composition in the duodenum and could be useful for further investigation of the duodenal microbiome.

Here, we establish a novel Sprague-Dawley (SD) rat model of superior sagittal sinus (SSS) thrombosis via a thread-embolization method, and the stability and reliability of the model were verified.

The functional genomic toolkit for the parasitic nematodes Strongyloides stercoralis and Strongyloides ratti includes transgenesis, CRISPR/Cas9-mediated mutagenesis, and RNAi. This protocol will demonstrate how to use intragonadal microinjection to introduce transgenes and CRISPR components into S. stercoralis and S. ratti.

Detailed protocol and three Python scripts are provided for operating an open-source robotic liquid handling system to perform semi-automated protein sample preparation for mass spectrometry experiments, covering detergent removal, protein digestion, and peptide desalting steps.

This work presents a flexible protocol for utilizing fluorescently labeled elastomeric contractible surfaces (FLECS) Technology in microwell format for simplified, hands-off quantification of single-cell contractile forces based on visualized displacements of fluorescent protein micropatterns.

This article demonstrates how to prepare and administer transferrin-bound nonradioactive isotopic iron for studies of iron transport in mouse pregnancy. The approach for quantifying isotopic iron in fetoplacental compartments is also described.

The present protocol describes new tools for SPR binding assays to examine CV-N binding to HA, S glycoprotein, related hybrid-type glycans, and high-mannose oligosaccharides. SPR is used to determine the K_D for binding either dimeric or monomeric CV-N to these glycans.

This protocol describes a novel method to deliver oral combinational antiretroviral drugs that successfully suppress HIV-1 RNA replication in humanized mice.

The present protocol describes an experimental platform to assess the effects of mechanical and biochemical cues on chemotherapeutic responses of patient-derived glioblastoma cells in 3D matrix-mimetic cultures using a custom-made UV illumination device facilitating high-throughput photocrosslinking of hydrogels with tunable mechanical features.

Here, we present a protocol for the intra-cardiac injection of human prostate cancer cells to generate a mouse model with bone metastasis lesions.

Utilizing an immunocompetent, autochthonous tumor model driven by common patient mutations for preclinical testing is critical for immunotherapeutic testing. This protocol describes a method to generate brain tumor mouse models using electroporation-based delivery of plasmid DNA that represent common patient mutations, thus providing an accurate, reproducible, and consistent mouse model.

This novel protocol entails the quantification of cardiovascular calcification progression from serial micro positron emission tomography (PET)/micro computed tomography (CT) images in small research animals.

We recently identified retinal capillary stiffening as a new paradigm for retinal dysfunction associated with diabetes. This protocol elaborates the steps for isolation of mouse retinal capillaries and the subendothelial matrix from retinal endothelial cultures, followed by a description of the stiffness measurement technique using atomic force microscopy.