655 results match your criteria Bio-protocol[Journal]


Isolation and Characterization of Exosomes from Mouse Feces.

Bio Protoc 2020 Apr;10(8)

Institute for Biomedical Sciences, Center for Diagnostics and Therapeutics, Digestive Disease Research Group, Georgia State University, Atlanta, Georgia, 30302, United States.

Exosomes secreted by colonic epithelial cells are present in feces and contain valuable epigenetic information, such as miRNAs, proteins, and metabolites. An in-depth study of this information is conducive to the diagnosis or treatment of relevant diseases. A crucial prerequisite of such a study is to establish an efficient isolation method, through which we can obtain a relatively more significant amount of exosomes from feces. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/bioprotoc.3584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7241525PMC

Optic Nerve Crush in Mice to Study Retinal Ganglion Cell Survival and Regeneration.

Bio Protoc 2020 Mar;10(6)

Department of Ophthalmology, Byers Eye Institute, Mary M. and Sash A. Spencer Center for Vision Research, Stanford University School of Medicine, Palo Alto, California 94034, USA.

In diseases such as glaucoma, the failure of retinal ganglion cell (RGC) neurons to survive or regenerate their optic nerve axons underlies partial and, in some cases, complete vision loss. Optic nerve crush (ONC) serves as a useful model not only of traumatic optic neuropathy but also of glaucomatous injury, as it similarly induces RGC cell death and degeneration. Intravitreal injection of adeno-associated virus serotype 2 (AAV2) has been shown to specifically and efficiently transduce RGCs and has thus been proposed as an effective means of gene delivery for the treatment of glaucoma. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3559DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7197875PMC

Protocol for Ribosome Profiling in Bacteria.

Bio Protoc 2019 Dec;9(24)

Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

Ribosome profiling provides information on the position of ribosomes on mRNA on a genomic scale. Although this information is often used to detect changes in gene expression under different conditions, it also has great potential for yielding insight into the mechanism and regulation of protein synthesis itself. First developed in yeast, ribosome profiling involves the isolation and sequencing of ribosome-protected mRNA fragments generated by nuclease treatment. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3468DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7079819PMC
December 2019

Laser Capture Micro-dissection (LCM) of Neonatal Mouse Forebrain for RNA Isolation.

Bio Protoc 2020 Jan;10(1)

Center for Integrative Brain Research, Seattle Children's Research Institute, Seattle, United States.

Precise and reproducible isolation of desired cell types or layers from heterogeneous tissues is crucial to analyze specific gene profiles and molecular interactions . Forebrain is the core site of higher functions, like cognition and memory consolidation. It is composed of heterogeneous and distinct cell types, interconnected to form functional neural circuits. Read More

View Article

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7079735PMC
January 2020

Steady-state and Flux-based Trehalose Estimation as an Indicator of Carbon Flow from Gluconeogenesis or Glycolysis.

Bio Protoc 2020 Jan;10(1):e3483

Institute for Stem Cell Science and Regenerative Medicine (inStem), GKVK Post Bellary Road, Bangalore 560065, India.

Trehalose (and glycogen) is a major storage carbohydrate in many cells, including . Typically, trehalose (a disaccharide of glucose) is synthesized and stored through gluconeogenesis. However, trehalose can also be made directly from glucose, if glucose-6-phosphate is channeled away from glycolysis or pentose phosphate pathway. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7075708PMC
January 2020

Analyzing the Functionality of Non-native Hsp70 Proteins in .

Bio Protoc 2019 Oct;9(19)

Department of Biological Sciences, The University of North Carolina at Charlotte, Charlotte, USA.

Yeast are an ideal system to study Heat Shock Protein 70 (Hsp70) function in a cellular context. This protocol was generated to analyze the function of non-native Hsp70 proteins by expressing them as the sole cytosolic Hsp70 in yeast. As an initial step, Hsp70 variants (such as Ssa1 point mutants and non-yeast versions such as NvHsp70A, B and D) are cloned into an appropriate expression plasmid. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7062368PMC
October 2019

Infection of Primary Human Monocytes with HIV-1.

Bio Protoc 2019 Jul;9(13)

Department of Microbiology and Molecular Genetics, College of Osteopathic Medicine, Michigan State University, East Lansing, USA.

Monocyte infection by HIV-1 is an important component of chronic HIV pathogenesis. Following infection by HIV-1, monocytes are able to cross the blood brain barrier and set up a viral reservoir in the central nervous system. Additionally, in the setting of chronic HIV-1 infection, monocytes can become activated either directly through HIV-1 infection or indirectly via HIV-1-mediated systemic immune activation. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/bioprotoc.3289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6990407PMC

Isolation and Culture of Murine Hepatic Stellate Cells.

Bio Protoc 2019 Nov;9(21)

Institute of Immunology and Infection Research, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3FL, UK.

Hepatic stellate cells (HSCs), alternatively known as liver pericytes, can differentiate into myofibroblasts and secrete extra-cellular matrix components, thereby promoting wound healing and fibrosis. Studying HSCs can provide insights into the pathological mechanisms governing these processes. HSC isolation methods typically comprise of enzymatic digestion followed by density gradient centrifugation and/or Fluorescent Activated Cell Sorting (FACS) mediated sorting. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3422DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6986919PMC
November 2019

3D Organoid Formation from the Murine Salivary Gland Cell Line SIMS.

Bio Protoc 2019 Oct;9(19)

Biointerfaces Institute, University of Michigan, Ann Arbor, MI 48109, USA.

Salivary glands consist of multiple phenotypically and functionally unique cell populations, such as the acinar, ductal, and myoepithelial cells that help produce, modify, and secrete saliva (Lombaert , 2011). Identification of mechanisms and factors that regulate these populations has been of key interest, as salivary gland-related diseases have detrimental effects on these cell populations. A variety of approaches have been used to understand the roles different signaling mechanisms and transcription factors play in regulating salivary gland development and homeostasis. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6953724PMC
October 2019

A Radioactive ERK3 Kinase Assay.

Bio Protoc 2019 Aug;9(16)

Department of Biochemistry and Molecular Biology, Wright State University, Dayton, OH, USA.

Mitogen-activated protein kinases (MAPKs) are serine/threonine kinases that have an important role in signal transduction. Extracellular signal-regulated kinase 3 (ERK3), also known as MAPK6, is an atypical MAPK. Here, we describe in detail an assay for the kinase activity of ERK3 using myelin basic protein (MBP) or steroid receptor coactivator-3 (SRC-3) as substrates. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3332DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6953734PMC

Glucosidase Inhibition to Study Calnexin-assisted Glycoprotein Folding in Cells.

Authors:
Hao Wang Qingyu Wu

Bio Protoc 2019 Jun;9(11)

Cyrus Tang Hematology Center, Collaborative Innovation Center of Hematology, State Key Laboratory of Radiation Medicine and Prevention, Soochow University, Suzhou, China.

Calnexin is a chaperone protein that plays a critical role in glycoprotein folding in the endoplasmic reticulum (ER). The function of calnexin depends on its binding to monoglucosylated oligosaccharides on nascent glycoproteins, whereas the generation of monoglucosylated oligosaccharides depends on the activity of α-glucosidases I and II, which trim off terminal glucose residues sequentially from triglucosylated N-glycans. This biochemical mechanism can be exploited to study calnexin-assisted folding and subsequent ER exiting of glycoproteins in cells. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6953735PMC

Isolation and Long-term Cultivation of Mouse Alveolar Macrophages.

Bio Protoc 2019 Jul;9(14)

Center for Regenerative Therapies Dresden (CRTD), Technische Universität Dresden, Fetscherstraße 105, Dresden, Germany.

Alveolar macrophages (AM) are tissue-resident macrophages that colonize the lung around birth and can self-maintain long-term in an adult organism without contribution of monocytes. AM are located in the pulmonary alveoli and can be harvested by washing the lungs using the method of bronchoalveolar lavage (BAL). Here, we compared different conditions of BAL to obtain high yields of murine AM for culture and expansion of AM. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6944498PMC

Semi-quantitative Determination of Protein Expression using Immunohistochemistry Staining and Analysis: An Integrated Protocol.

Bio Protoc 2019 Dec;9(24)

Department of Pharmaceutical Sciences, College of Pharmacy, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.

Semi-quantitative IHC is a powerful method for investigating protein expression and localization within tissues. The semi-quantitative immunohistochemistry (IHC) involves using software such as free software ImageJ Fiji to conduct deconvolution and downstream analysis. Currently, there is lack of an integrated protocol that includes a detailed procedure of how to measure or compare protein expression. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6924920PMC
December 2019

Intestinal Epithelial Wound-healing Assays Using Electric Cell-Substrate Impedance Sensing Instrument.

Bio Protoc 2019 Sep;9(17)

Institute for Biomedical Sciences, Center for Diagnostics and Therapeutics, Digestive Disease Research Group, Georgia State University, Atlanta, 30302, USA.

Here, we describe an epithelial wound-healing assay using Electric Cell-Substrate Impedance Sensing (ECIS) technology. The ECIS technology is a real time cell growth assay based on a small (250 μm diameter) active gold electrode which resistance is measured continuously. When intestinal epithelial cells reach confluency on the gold electrode, resistances reach a plateau. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6910258PMC
September 2019

Genotyping of the OATP1B1 c. 521 T>C Polymorphism from the Formalin-Fixed Paraffin-Embedded (FFPE) Tissue Specimens: an Optimized Protocol.

Bio Protoc 2019 Aug;9(16)

1110 N. Stonewall Avenue, Department of Pharmaceutical Sciences, College of Pharmacy, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.

Organic anion transporting polypeptide (OATP) 1B1 is a liver-specific transport protein that plays an important role in hepatic drug disposition. It transports many drugs from the blood into the liver, including lipid-lowering statins. The c. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6909936PMC

High Throughput Traction Force Microscopy for Multicellular Islands on Combinatorial Microarrays.

Bio Protoc 2019 Nov;9(21)

Department of Bioengineering, University of Illinois Urbana Champaign, Champaign, IL, USA.

The composition and mechanical properties of the cellular microenvironment along with the resulting distribution of cellular devolved forces can affect cellular function and behavior. Traction Force Microscopy (TFM) provides a method to measure the forces applied to a surface by adherent cells. Numerous TFM systems have been described in literature. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6897381PMC
November 2019

Antisense Oligodeoxynucleotide Perfusion Blocks Gene Expression of Synaptic Plasticity-related Proteins without Inducing Compensation in Hippocampal Slices.

Bio Protoc 2019 Oct;9(19)

Department of Physiology and Pharmacology, The Robert F. Furchgott Center for Neural and Behavioral Science, State University of New York Downstate Medical Center, Brooklyn, United States.

The elucidation of the molecular mechanisms of long-term synaptic plasticity has been hindered by both the compensation that can occur after chronic loss of the core plasticity molecules and by conditions that may not reproduce plasticity. Here we describe a novel method to rapidly suppress gene expression by antisense oligodeoxynucleotides (ODNs) applied to rodent brain slices in an "Oslo-type" interface chamber. The method has three advantageous features: 1) rapid blockade of new synthesis of the targeted proteins that avoids genetic compensation, 2) efficient oxygenation of the brain slice, which is critical for reproducing conditions of long-term synaptic plasticity, and 3) a recirculation system that uses only small volumes of bath solution (< 5 ml), reducing the amount of reagents required for long-term experiments lasting many hours. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6892586PMC
October 2019

Sciatic Nerve Cut and Repair Using Fibrin Glue in Adult Mice.

Bio Protoc 2019 Sep;9(18)

Department of Physiology, Emory University, Atlanta, USA.

Peripheral nerve injury (PNI) is an excellent model for studying neural responses to injury and elucidating the mechanisms that can facilitate axon regeneration. As such, several animal models have been employed to study regenerative mechanisms after PNI, including Aplysia, zebrafish, rabbits, cats and rodents. This protocol describes how to perform a sciatic nerve injury and repair in mice, one of the most frequently used models to study mechanisms that facilitate recovery after PNI, and that takes advantage of the availability of many genetic models. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6884152PMC
September 2019

Isolation, Purification, and Characterization of Ginger-derived Nanoparticles (GDNPs) from Ginger, Rhizome of .

Bio Protoc 2019 Oct;9(19)

Institute for Biomedical Science, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA, USA.

Factors implicated in the pathophysiology of intestinal inflammation include defects in intestinal epithelial barrier function, abnormal immune responses, and activities of the gut microbiota. Current agents used to treat human Inflammatory Bowels Disease (IBD), chronic inflammation of digestive tract, have serious side effects. In addition, most of these treatments target the damaging factors while not providing pro-healing factors that repair the damaged intestine. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6857645PMC
October 2019

Adhesive and Cohesive Peel Force Measurement of Human Airway Mucus.

Bio Protoc 2019 Jul;9(13)

Marsico Lung Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7248, USA.

In health, the high-speed airflow associated with cough represents a vital backup mechanism for clearing accumulated mucus from our airways. However, alterations in the mucus layer in cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) leads to the mucus layer adhered to the airway surfaces, representing the nidus of chronic lung infection. To understand what is different about diseased mucus and why cough clearance is defective, there is a need for techniques to quantify the strength of the interactions limiting the ability of airflow to strip mucus from the airway surface (. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6827558PMC
July 2019
1 Read

Quantitation of TLR4 Internalization in Response to LPS in Thioglycollate Elicited Peritoneal mouse Macrophages by Flow Cytometry.

Bio Protoc 2019 Sep;9(18)

Department of Microbiology and Immunology, University of Maryland Baltimore, Baltimore, USA.

Internalization of cell surface Toll Like Receptor 4 (TLR4) is a convenient read-out to measure LPS dependent activation of the TRIF adaptor pathway. We here provide a protocol to quantify the LPS dependent internalization of TLR4 using thioglycollate-elicited peritoneal macrophages by flow cytometry. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3369DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6812529PMC
September 2019

A High-throughput and Pathophysiologically Relevant Astrocyte-motor Neuron Co-culture Assay for Amyotrophic Lateral Sclerosis Therapeutic Discovery.

Bio Protoc 2019 Sep;9(17)

Sheffield Institute for Translational Neuroscience (SITraN), University of Sheffield, Sheffield, S10 2HQ, UK.

Amyotrophic lateral sclerosis (ALS) is an adult onset neurological disorder characterized by loss of motor neurons leading to progressive muscle wasting and eventually death. Astrocytes play a key role in disease pathogenesis. However, the ability to study astrocytic support towards motor neurons in ALS has been limited by a lack of sustainable high-throughput human cell models. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3353DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6774773PMC
September 2019
1 Read

Fibroblast Gap-closure Assay-Microscopy-based Assay Measuring the Migration of Murine Fibroblasts.

Bio Protoc 2019 Aug;9(16)

Division of Pulmonary, Critical Care, Allergy and Sleep Medicine, Department of Medicine, University of California, San Francisco; CA 94158, USA.

Pulmonary fibrosis is characterized by pathological scaring of the lung. Similar to other fibrotic diseases, scar formation is driven by excessive extracellular matrix deposition by activated, proliferative, and migratory fibroblasts. Currently, the two most widely used chemotaxis and cell migration assays are the scratch assay and the transmembrane invasion assay. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3333DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6748635PMC

Total RNA Isolation from Separately Established Monolayer and Hydrogel Cultures of Human Glioblastoma Cell Line.

Bio Protoc 2019 Jul;9(14)

Department of Biology, New Mexico State University, Las Cruces, NM, USA.

Astrocytoma is an invasive carcinoma occurring in the nervous system and currently lacks effective treatment options. A deeper understanding of the mechanisms of tumorigenesis and tumor progression is needed in order to develop novel therapeutic strategies. Recent advances in culture systems have demonstrated that the use of three-dimensional (3D) culture models could be more relevant for this purpose as compared to monolayer or two-dimensional (2D) models due to their resemblance to cancer pathology. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6746428PMC
July 2019
1 Read

Cross-linking, Immunoprecipitation and Proteomic Analysis to Identify Interacting Proteins in Cultured Cells.

Bio Protoc 2019 Jun;9(11)

Department of Cardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, USA.

Extracellular expression is essential for the function of secreted and cell surface proteins. Proper intracellular trafficking depends on protein interactions in multiple subcellular compartments. Co-immunoprecipitation and the yeast two-hybrid system are commonly used to investigate protein-protein interactions. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3258DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6746336PMC

Precision Technique for Splenectomy Limits Mouse Stress Responses for Accurate and Realistic Measurements for Investigating Inflammation and Immunity.

Bio Protoc 2019 Aug;9(15)

Pediatric Surgery, Children's Hospital of Orange County, Orange, CA 92868-3874, United States of America.

Splenectomy in an animal model requires a standardized technique utilizing best practice to avoid variability which can result in adverse impact to the animal resulting in flawed physiologic responses simply due to technique rather than to the studied variables. In the case of the spleen, often investigators are analyzing the animal immune or inflammatory responses. Surgical splenectomy involves many variables from the training and expertise of the surgeon, which directly correlates to surgical technique to the length of operation and ease of the procedure. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6730662PMC
August 2019
2 Reads

Visualization of Macropinocytosis in Prostate Fibroblasts.

Bio Protoc 2019 May;9(10)

Hematology/Oncology, Cedars-Sinai Medical Center, Los Angeles, USA.

Macropinocytosis has emerged as an important mechanism for non-selective route to internalize extracellular fluids and dissolved molecules in eukaryotic cell. As fundamental cellular behavior, macropinocytosis plays specific and distinct roles in many physiological and pathological processes, such as nutrients uptake, antigen presentation, pathogen capture, and tumorigenesis. It supports tumorigenesis by providing metabolic needs to dividing cells in Ras driven cancer. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6716514PMC
May 2019
1 Read

Inducing Alcohol Dependence in Rats Using Chronic Intermittent Exposure to Alcohol Vapor.

Bio Protoc 2019 May;9(9)

Department of Physiology, Louisiana State University Health Science Center, New Orleans, LA 70112, USA.

Alcohol use disorder (AUD) is a significant public health and economic burden and is often characterized by repeated bouts of alcohol intoxication and withdrawal. Virtually all organ systems are impacted by chronic alcohol exposure. These effects can be investigated using the rat as a model organism; however, rats typically will not self-administer alcohol to levels necessary to achieve physiological and behavioral aspects of dependence. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6699762PMC

Northern Blot with IR Fluorescent Probes: Strategies for Probe Preparation.

Bio Protoc 2019 Apr;9(8)

Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, United States.

Northern blot is a molecular biology technique that can detect, quantify, and determine the molecular weight of RNA. Recently, we published a protocol utilizing near-infrared (IR) fluorescent probes in Northern blot (irNorthern). Our method is as sensitive as other non-radioactive methods but is more straightforward and versatile. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6699753PMC
April 2019
1 Read

TZA, a Sensitive Reporter Cell-based Assay to Accurately and Rapidly Quantify Inducible, Replication-competent Latent HIV-1 from Resting CD4 T Cells.

Bio Protoc 2019 May;9(10)

Infectious Diseases and Microbiology, University of Pittsburgh, Pittsburgh, USA.

The latent HIV-1 viral reservoir in resting CD4 (rCD4) T cells represents a major barrier to an HIV-1 cure. There is an ongoing effort to identify therapeutic approaches that will eliminate or reduce the size of this reservoir. However, clinical investigators lack an assay to determine whether or not a decrease in the latent reservoir has been achieved. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6699756PMC
May 2019
1 Read

Triple Fluorescence Anisotropy Reporter Imaging in Living Cells.

Bio Protoc 2019 May;9(9)

Department of Physiology, University of Maryland Baltimore, Maryland, United States.

FRET-based genetically encoded biosensors incorporate two fluorescent proteins into their design to enable ratiometric biosensing of signaling activities in live cells. While emission ratios are generally useful for quantitative studies, they leave little room in the optical spectrum for additional sensors and optogenetic tools. Homotransfer-based reporters, such as the FLuorescence Anisotropy REporters (FLAREs), incorporate two fluorescent proteins of the same color into their design. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697128PMC

Optical Stimulation and Electrophysiological Analysis of Regenerating Peripheral Axons.

Bio Protoc 2019 Jun;9(12)

Department of Cell Biology, Emory University School of Medicine, Atlanta, GA, USA.

Although axons in the peripheral nervous system can regenerate, functional recovery after nerve injuries is poor. Activity-based therapies, such as exercise and electrical stimulation, enhance the regeneration of cut peripheral axons. Despite their effectiveness, clinical application of these experimental techniques has been limited. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6688771PMC
June 2019
4 Reads

Visualizing Filamentous Actin Using Phalloidin in .

Bio Protoc 2019 Jun;9(12)

Anatomy & Cell Biology, University of Kansas Medical Center, Kansas City, KS, USA.

This protocol aims to visualize the filamentous actin network in . We improved fixed-cell labeling conditions using the F-actin probe, phalloidin. We created a -optimized protocol by halving the phalloidin incubation time, electing for optimal fixation conditions, and selecting for a healthy cell population. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667228PMC

Enterovirus Competition Assay to Assess Replication Fitness.

Bio Protoc 2019 Jun 20;9(10). Epub 2019 May 20.

Department of Pathology, University of Cambridge, Cambridge, UK.

In virology the difference between the fitness of two viruses can be determined by using various methods, such as virus titer, growth curve analysis, measurement of virus infectivity, analysis of produced RNA copies and viral protein production. However, for closely performing viruses, it is often very hard to distinguish the differences. competition assays are a sensitive tool for determining viral replication fitness for many viruses replicating in cell culture. Read More

View Article

Download full-text PDF

Source
https://bio-protocol.org/e3233
Publisher Site
http://dx.doi.org/10.21769/BioProtoc.3233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6561778PMC
June 2019
6 Reads

Correction: Polysome Fractionation to Analyze mRNA Distribution Profiles.

Bio Protoc 2019 Feb;9(3)

Laboratory of Genetics and Genomics, National Institute on Aging, National Institutes of Health, Biomedical Research Center, Baltimore, USA.

[This corrects the article PMC5431591.]. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6482963PMC
February 2019
10 Reads

Flip-flop Mediated Conditional Gene Inactivation in .

Bio Protoc 2019 Feb;9(3)

Centre for Cellular & Molecular Biology, Hyderabad, India.

Mosaic analysis in , an important tool to assess cellular phenotypes of mutants in an otherwise heterozygous background, relies on mitosis. Hence, it cannot be used to inactivate gene function in mitotically inactive, terminally differentiated cells such as neurons. To address this issue, we developed "Flip-flop", a novel, Flippase-dependent cassette-inversion method that functions independent of mitosis, and therefore can be used for gene inactivation in both mitotic as well as postmitotic cells. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6553656PMC
February 2019
1 Read

Non-radiometric Cell-free Assay to Measure the Effect of Molecular Chaperones on AMP-activated Kinase Activity.

Bio Protoc 2019 Apr;9(8)

McAllister Heart Institute, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

AMP-activated kinase (AMPK) is a trimeric protein holoenzyme with kinase activity. AMPK plays an important role in cellular metabolism and is thought to function as a fuel sensor within the cell, exerting kinase activity to activate energy-conserving pathways and simultaneously inhibit energy-consuming pathways. Traditional methods to measure AMPK activity to test potential agonists or antagonists utilize radiolabeled ATP with a peptide substrate. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6534159PMC
April 2019
1 Read

Preparation and Manipulation of Olfactory Epithelium Explant Cultures for Measurement of the Mechanical Tension of Individual Axons Using the Biomembrane Force Probe.

Bio Protoc 2019 Apr;9(8)

Sorbonne Université, CNRS, INSERM, Institut de Biologie Paris Seine, Laboratoire Neuroscience Paris Seine, F-75005 Paris, France.

In this paper, we describe a protocol allowing measurement of the mechanical tension of individual axons grown from neural tissue explants. This protocol was developed with primary cultures of olfactory epithelium explants from embryonic (E13.5) mice. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6534158PMC
April 2019
15 Reads

Identification of RNase-sensitive LINE-1 Ribonucleoprotein Interactions by Differential Affinity Immobilization.

Bio Protoc 2019 Apr;9(7)

Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, USA.

Long Interspersed Nuclear Element-1 (LINE-1, L1) constitutes a family of autonomous, self-replicating genetic elements known as retrotransposons. Although most are inactive, copious L1 sequences populate the human genome. L1s proliferate in a 'copy-and-paste' fashion through an RNA intermediate; a full-length L1 transcript is ~6,000 nucleotides long and functions as a bicistronic mRNA that encodes and assembles with two main polypeptides, ORF1p and ORF2p, forming a ribonucleoprotein (RNP); L1 RNPs also interact with a wide range of host factors in positive and negative regulatory capacities. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6519465PMC
April 2019
1 Read

Biofilm Assays on Fibrinogen-coated Silicone Catheters and 96-well Polystyrene Plates.

Bio Protoc 2019 Mar;9(6)

Department of Biological Sciences, University of Notre Dame, Notre Dame, IN, USA.

Biofilm formation is a well-known bacterial strategy that protects cells from hostile environments. During infection, bacteria found in a biofilm community are less sensitive to antibiotics and to the immune response, often allowing them to colonize and persist in the host niche. Not surprisingly, biofilm formation on medical devices, such as urinary catheters, is a major problem in hospital settings. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6519467PMC
March 2019
8 Reads

Primary Embryonic Rat Cortical Neuronal Culture and Chronic Rotenone Treatment in Microfluidic Culture Devices.

Bio Protoc 2019 Mar;9(6)

University of Pittsburgh Department of Neurology, University of Pittsburgh, Pittsburgh, PA 15213, USA.

In the study of neurodegenerative diseases, it is imperative to study the cellular and molecular changes associated with pathogenesis in the relevant cell type, central nervous system neurons. The unique compartmentalized morphology and bioenergetic needs of primary neurons present complications for their study in culture. Recent microculture techniques utilizing microfluidic culture devices allows for environmental separation and analysis of neuronal cell bodies and neurites in culture. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6519463PMC
March 2019
2 Reads

Murine Pharmacokinetic Studies.

Bio Protoc 2018 Oct;8(20)

Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, The Ohio State University, Columbus, OH, USA.

Murine pharmacokinetics (PK) represents the absorption, distribution, metabolism, and elimination of drugs from the body, which helps to guide clinical studies, ultimately resulting in more effective drug treatment. The purpose of this protocol is to describe a serial bleeding protocol, obtaining blood samples at six time points from single mouse to yield a complete PK profile. This protocol has proved to be rapid, highly repeatable, and relatively easy to acquire. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6521687PMC
October 2018
2 Reads

Viral Chromosome Conformation Capture (V3C) Assays for Identifying Trans-interaction Sites between Lytic Viruses and the Cellular Genome.

Bio Protoc 2019 Mar;9(6)

Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Columbia, Missouri, USA.

The folding mechanisms of the mammalian genome package our genetic material into the nucleus, and in doing so, dictate its appropriate replication and expression. Chromosome conformation capture technology has enabled the dissection of the folding principles of the cellular genome. This has led to a better understanding of the role played by architectural proteins in forming and dissolving 3D-chromatin-structure. Read More

View Article

Download full-text PDF

Source
https://bio-protocol.org/e3198
Publisher Site
http://dx.doi.org/10.21769/BioProtoc.3198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6482961PMC
March 2019
7 Reads

Single-cell Motility Analysis of Tethered Human Spermatozoa.

Bio Protoc 2019 Mar;9(5)

Department of Urology, University of California, San Francisco, CA, USA.

Vigorous sperm flagellar motility is essential for fertilization, and so the quantitative measurement of motility is a useful tool to assess the intrinsic fertility potential of sperm cells and explore how various factors can alter sperm's ability to reach the egg and penetrate its protective layers. Human sperm beat their flagella many times each second, and so recording and accurately quantifying this movement requires a high-speed camera. The aim of this protocol is to provide a detailed description of the tools required for quantitative beat frequency measurement of tethered human sperm at the single-cell level and to describe methods for investigating the effects of intracellular or extracellular factors on flagellar motion. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3182DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6482966PMC
March 2019
1 Read

Infection Model of Murine Small Intestine.

Bio Protoc 2018 Dec;8(24)

Instituto de Medicina Molecular | João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal.

is a tissue specific, intracellular protozoan that infects the murine small intestinal epithelia, which has been widely used as a coccidian model to study mucosal immunology. This mouse infection model is valuable to investigate the mechanisms of host protection against primary and secondary infection in the small intestine. Here, we describe the generation of an stock solution, preparation of sporulated to infect mice and determination of oocysts burden. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6485400PMC
December 2018

Papain-based Single Cell Isolation of Primary Murine Brain Endothelial Cells Using Flow Cytometry.

Bio Protoc 2018 Nov;8(22)

Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA, USA.

Brain endothelial cells (BECs) form the integral component of the blood-brain barrier (BBB) which separates the systemic milieu from the brain parenchyma and protects the brain from pathogens and circulating factors. In order to study BEC biology, it was of particular interest to establish a method that enables researchers to investigate and understand the underlying molecular mechanisms regulating their function during homeostasis, aging and disease. Furthermore, due to the heterogeneity of the cerebrovasculature and different vessel types that comprise the BBB, it is of particular interest to isolate primary BECs for single cell analysis from various subregions of the brain, such as the neurogenic and highly vascularized hippocampus and to enrich for specific vessel types. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6482965PMC
November 2018
1 Read

Preparation of RNA 3' End Sequencing Libraries of Total and 4-thiouracil Labeled RNA for Simultaneous Measurement of Transcription, RNA Synthesis and Decay in .

Bio Protoc 2019 Mar;9(6)

Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.

Cellular RNA levels are determined by the rates of RNA transcription from the gene template and subsequent RNA stability. Knowledge about both transcription and RNA decay is, therefore, necessary to interpret RNA levels and gene expression, especially during cellular processes where these parameters change. Numerous experimental strategies have been developed to measure transcription and RNA decay rates. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6436697PMC
March 2019
3 Reads

Characterization of Mouse Adult Testicular Macrophage Populations by Immunofluorescence Imaging and Flow Cytometry.

Bio Protoc 2019 Mar;9(5)

Aix Marseille Université, CNRS, INSERM, CIML, Marseille, France.

Testicular macrophages (tMΦ) are the most abundant immune cells residing in the testis, an immune-privileged organ. TMΦ are known to exhibit different functions, such as protecting spermatozoa from auto-immune attack by producing immunosuppressive cytokines and trophic roles in supporting spermatogenesis and male sex hormone production. They also contribute to fetal testicular development. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.3178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6438457PMC
March 2019
13 Reads

Preserve Cultured Cell Cytonemes through a Modified Electron Microscopy Fixation.

Bio Protoc 2018 Jul;8(13)

Department of Cell and Molecular Biology, St. Jude Children's Hospital, Memphis, TN, USA.

Immunocytochemistry of cultured cells is a common and effective technique for determining compositions and localizations of proteins within cellular structures. However, traditional cultured cell fixation and staining protocols are not effective in preserving cultured cell cytonemes, long specialized filopodia that are dedicated to morphogen transport. As a result, limited mechanistic interrogation has been performed to assess their regulation. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.2898DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6426145PMC
July 2018
1 Read

Hypoxia Reporter Element Assay.

Bio Protoc 2018 Aug;8(15)

Division of Pharmaceutics, College of Pharmacy and Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, USA.

Hypoxia is a condition in which there is a decrease in oxygen supply to the cellular environment. Changes to cellular oxygen levels can lead to transcriptional changes of oxygen-regulated genes. Reporter assays are used to study gene expression alteration and modifications in response to environmental changes. Read More

View Article

Download full-text PDF

Source
http://dx.doi.org/10.21769/BioProtoc.2951DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6424511PMC
August 2018
1 Read