602 results match your criteria Bio-protocol[Journal]


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

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

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

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http://dx.doi.org/10.21769/BioProtoc.3178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6438457PMC
March 2019
2 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

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

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

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

On-demand Labeling of SNAP-tagged Viral Protein for Pulse-Chase Imaging, Quench-Pulse-Chase Imaging, and Nanoscopy-based Inspection of Cell Lysates.

Bio Protoc 2019 Feb;9(4)

School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom.

Advanced labeling technologies allow researchers to study protein turnover inside intact cells and to track the labeled protein in downstream applications. In the context of a viral infection, the combination of imaging and fluorescent labeling of viral proteins sheds light on their biological activity and interaction with the host cell. Initial approaches have fused fluorescent proteins such as green fluorescent protein (GFP) to the viral protein-of-interest. Read More

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http://dx.doi.org/10.21769/BioProtoc.3177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6420817PMC
February 2019

Imaging Higher-order Chromatin Structures in Single Cells Using Stochastic Optical Reconstruction Microscopy.

Bio Protoc 2019 Feb;9(3)

Biomedical Optical Imaging Laboratory, Departments of Medicine and Bioengineering, University of Pittsburgh, Pittsburgh, PA 15213, USA.

Higher-order chromatin organization shaped by epigenetic modifications influence the chromatin environment and subsequently regulate gene expression. Direct visualization of the higher-order chromatin structure at their epigenomic states is of great importance for understanding chromatin compaction and its subsequent effect on gene expression and various cellular processes. With the recent advances in super-resolution microscopy, the higher-order chromatin structure can now be directly visualized down to the scale of ~30 nm. Read More

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http://dx.doi.org/10.21769/BioProtoc.3160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6414075PMC
February 2019

A Mouse Model of Postoperative Pain.

Bio Protoc 2019 Jan;9(2)

Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, WI 53226, USA.

Postoperative pain is highly debilitating and hinders recovery. Opioids are the main pain medication used for acute postoperative pain. Given the devastating opioid addiction and overdose epidemic across the US, non-opioid pain therapeutics are desperately needed. Read More

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http://dx.doi.org/10.21769/BioProtoc.3140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6390978PMC
January 2019
1 Read

Assessing Yeast Cell Survival Following Hydrogen Peroxide Exposure.

Bio Protoc 2019 Jan;9(2)

Department of Biological Sciences, UMBC, Baltimore, MD 21250, USA.

In the presence of oxidative stress, cellular defense systems that can detoxify reactive oxygen species are activated through multiple signaling cascades and transcriptional reprogramming. The budding yeast has served as an excellent model for genetically-identifying factors important for the response to oxidative stress. Here, we describe two assays for testing yeast gene deletion strains or strains overexpressing a gene of interest for viability following oxidative stress induced by hydrogen peroxide treatment. Read More

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http://dx.doi.org/10.21769/BioProtoc.3149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6385872PMC
January 2019

Heterochronic Phenotype Analysis of Hypodermal Seam Cells in .

Authors:
Yon Ju Ji Jiou Wang

Bio Protoc 2019 01;9(1)

Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA.

Heterochrony refers to changes in the timing of developmental events, and it is precisely regulated in the organisms by the heterochronic genes such as and . Mutations in these genes cause precocious or retarded development of certain cell lineages. With well-defined cell lineages, is one of the best model systems to study heterochronic genes, since the subtle changes in the development of cell lineages can be easily identified. Read More

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http://dx.doi.org/10.21769/BioProtoc.3132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6368992PMC
January 2019

Estimation of the Readily Releasable Synaptic Vesicle Pool at the Larval Neuromuscular Junction.

Bio Protoc 2019 Jan;9(1)

Department of Neurobiology, University of Southern California, Los Angeles, USA.

Presynaptic boutons at nerve terminals are densely packed with synaptic vesicles, specialized organelles for rapid and regulated neurotransmitter secretion. Upon depolarization of the nerve terminal, synaptic vesicles fuse at specializations called active zones that are localized at discrete compartments in the plasma membrane to initiate synaptic transmission. A small proportion of synaptic vesicles are docked and primed for immediate fusion upon synaptic stimulation, which together comprise the readily releasable pool. Read More

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http://dx.doi.org/10.21769/BioProtoc.3127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6370317PMC
January 2019
1 Read

Blinded Visual Scoring of Images Using the Freely-available Software Blinder.

Bio Protoc 2018 Dec;8(23)

Nicholas School of the Environment, Duke University, Durham, NC, USA.

In nearly all subfields of biomedical sciences, there are phenotypes that are currently classified by expert visual scoring. In research applications, these classifications require the experimenter to be blinded to the treatment group in order to avoid unintentional bias in scoring. Currently, many labs either use laborious and tedious methods to manually blind the images, require multiple experimenters to gather and score the data blindly or fail to properly blind the data altogether. Read More

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http://dx.doi.org/10.21769/BioProtoc.3103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6370323PMC
December 2018

An Microscopy-based Assay for Microtubule-binding and Microtubule-crosslinking by Budding Yeast Microtubule-associated Protein.

Bio Protoc 2018 Dec;8(23)

Department of Biological Sciences, Dartmouth College, Hanover, NH 03755, USA.

In this protocol, we describe a simple microscopy-based method to assess the interaction of a microtubule-associated protein (MAP) with microtubules. The interaction between MAP and microtubules is typically assessed by a co-sedimentation assay, which measures the amount of MAP that co-pellets with microtubules by centrifugation, followed by SDS-PAGE analysis of the supernatant and pellet fractions. However, MAPs that form large oligomers tend to pellet on their own during the centrifugation step, making it difficult to assess co-sedimentation. Read More

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http://dx.doi.org/10.21769/BioProtoc.3110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6363367PMC
December 2018
1 Read

Analysis of the Mitochondrial Membrane Potential Using the Cationic JC-1 Dye as a Sensitive Fluorescent Probe.

Bio Protoc 2019 Jan;9(1)

Department of Pharmaceutical Sciences, Texas Tech University Health Sciences Center, Amarillo, TX 79106, USA.

In recent years, fluorescent dyes have been frequently used for monitoring mitochondrial membrane potential to evaluate mitochondrial viability and function. However, the reproducibility of the results across laboratories strongly depends upon following well validated and reliable protocols along with the appropriate controls. Herein, we provide a practical user guide for monitoring mitochondrial membrane potential in whole cells using a fluorescent cationic probe. Read More

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http://dx.doi.org/10.21769/BioProtoc.3128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6343665PMC
January 2019

A Functionally Robust Phenotypic Screen that Identifies Drug Resistance-associated Genes Using 3D Cell Culture.

Bio Protoc 2018 Nov;8(22)

Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.

Drug resistance is a major obstacle in cancer treatment: A case in point is the failure of cancer patients to respond to tyrosine kinase inhibitors (TKI) of EGFR, a receptor that is highly expressed in many cancers. Identification of the targets and delineation of mechanisms of drug resistance remain major challenges. Traditional pharmacological assays of drug resistance measure the response of tumor cells using cell proliferation or cell death as readouts. Read More

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http://dx.doi.org/10.21769/BioProtoc.3083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6347116PMC
November 2018
2 Reads

Eye Drops for Delivery of Bioactive Compounds and BrdU to Stimulate Proliferation and Label Mitotically Active Cells in the Adult Rodent Retina.

Bio Protoc 2018 Nov;8(21)

Department of Biological Sciences, Western Michigan University, Kalamazoo, MI, USA.

Eye drop treatments are typically used to apply drugs to the anterior structures of the eye. Recently, however, studies have demonstrated that eye drops can reach the retina in the back of the eye if pharmacological agents are carried in appropriate vehicles. Here, we introduce an eye drop procedure to deliver a drug (PNU-282987), in combination with BrdU, to stimulate cell cycle re-entry and label dividing cells in the retinas of adult rodents. Read More

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http://dx.doi.org/10.21769/BioProtoc.3076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6347112PMC
November 2018

Detection of Ligand-binding to Membrane Proteins by Capacitance Measurements.

Bio Protoc 2019 01;9(1)

Institute of Pharmacology and the Gaston H. Glock Research Laboratories for Exploratory Drug Development, Center of Physiology and Pharmacology, Medical University of Vienna, Vienna, Waehringerstrasse 13a, A-1090 Vienna, Austria.

In multi-cellular organisms, cells communicate with each other utilizing chemical messengers. For many of these messenger molecules, the membrane is an insurmountable barrier. Yet, they act by binding to surface proteins often triggering a cascade of reactions inside the cell. Read More

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http://dx.doi.org/10.21769/BioProtoc.3138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6342252PMC
January 2019
1 Read

Microirradiation for Precise, Double-strand Break Induction in .

Bio Protoc 2018 Dec;8(24)

Department of Biology, University of Iowa, Iowa City, USA.

DNA double-strand breaks (DSBs) are toxic lesions that every cell must accurately repair in order to survive. The repair of DSBs is an integral part of a cell life cycle and can lead to lethality if repaired incorrectly. Laser microirradiation is an established technique which has been used in yeast, mammalian cell culture, and cell culture to study the regulation of DSB repair. Read More

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https://bio-protocol.org/e3130
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http://dx.doi.org/10.21769/BioProtoc.3130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6342474PMC
December 2018
12 Reads

Generation of CRISPR-Cas9 Complexes with Covalently Bound Repair Templates for Genome Editing in Mammalian Cells.

Bio Protoc 2019 Jan;9(1)

The Institute of Molecular Health Sciences, ETH Zurich, Zurich, Switzerland.

The CRISPR-Cas9 system is a powerful genome-editing tool that promises application for gene editing therapies. The Cas9 nuclease is directed to the DNA by a programmable single guide (sg)RNA, and introduces a site-specific double-stranded break (DSB). In mammalian cells, DSBs are either repaired by non-homologous end joining (NHEJ), generating small insertion/deletion (indel) mutations, or by homology-directed repair (HDR). Read More

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https://bio-protocol.org/e3136
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http://dx.doi.org/10.21769/BioProtoc.3136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6340478PMC
January 2019
4 Reads

Quantification of Mouse Hematopoietic Progenitors' Formation Using Time-lapse Microscopy and Image Analysis.

Bio Protoc 2019 01;9(1)

European Molecular Biology Laboratory, EMBL Rome, Epigenetics and Neurobiology Unit Monterotondo, Italy.

differentiation of mouse embryonic stem cells (mESCs) towards blood cells constitutes a well-established system to study the endothelial-to-hematopoietic transition (EHT) at the onset of blood development. Assessing the emergence of small non-adherent round blood cells in the culture without disturbing it is essential to evaluate the progression of EHT and also to test conditions potentially enhancing or repressing this process. Here, we describe how to quantify the formation of mouse hematopoietic progenitors during EHT in normal conditions or following over-expression of eight essential transcription factors using time-lapse microscopy and image analysis. Read More

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http://dx.doi.org/10.21769/BioProtoc.3137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6331043PMC
January 2019
1 Read

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

Bio Protoc 2018 Nov;8(22)

Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.

The brain endothelium is a highly specialized vascular structure that maintains the activity and integrity of the central nervous system (CNS). Previous studies have reported that the integrity of the brain endothelium is compromised in a plethora of neuropathologies. Therefore, it is of particular interest to establish a method that enables researchers to investigate and understand the molecular changes in CNS endothelial cells and underlying mechanisms in conjunction with murine models of disease. Read More

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http://dx.doi.org/10.21769/BioProtoc.3092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6326170PMC
November 2018
1 Read

Method for Studying the Effect of Gene Silencing on Bacterial Infection-induced ERK1/2 Signaling in Bone-marrow Derived Macrophages.

Bio Protoc 2018 Dec;8(24)

Division of Cell Biology and Immunology, CSIR-Institute of Microbial Technology (IMTECH), Chandigarh, India.

Macrophages are highly phagocytic cells that utilize various pathogen recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs). These PAMPs can be present within the microbe, such as bacterial CpG DNA, and are recognized by Toll-like receptor 9 (TLR9), a PRR present on the endosomal membrane of macrophages. PAMPs can also be present on the surface of microbes, such as Lipopolysaccharide (LPS), which decorates the outer membrane of gram-negative bacteria like and . Read More

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http://dx.doi.org/10.21769/BioProtoc.3123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6322648PMC
December 2018
1 Read

Mosaic Labeling and 3-Dimensional Morphological Analysis of Single Cells in the Zebrafish Left-right Organizer.

Bio Protoc 2018 Nov;8(22)

Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, NY, USA.

A transient epithelial structure called the left-right organizer (LRO) establishes left-right asymmetry in vertebrate embryos. Developmental defects that alter LRO formation result in left-right patterning errors that often lead to congenital heart malformations. However, little is known about mechanisms that regulate individual cell behaviors during LRO formation. Read More

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http://dx.doi.org/10.21769/BioProtoc.3090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6319891PMC
November 2018

Vascular Permeability Assay in Human Coronary and Mouse Brachiocephalic Arteries.

Bio Protoc 2018 Oct;8(20)

CVPath Institute, Gaithersburg, Maryland, USA.

Coronary artery disease remains an important cause of morbidity and mortality. Previous work, including ours, has focused on the role of intraplaque hemorrhage, particularly from immature microvessel angiogenesis, as an important contributor to plaque progression via increases in vascular permeability leading to further intraplaque hemorrhage, which increases red cell membrane-derived free cholesterol in plaque content and inflammatory cell recruitment. Evans Blue Dye (EBD) assay is widely used as a standard assay for vasculature permeability. Read More

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https://bio-protocol.org/e3048
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http://dx.doi.org/10.21769/BioProtoc.3048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6319945PMC
October 2018
11 Reads

Notochord Injury Assays that Stimulate Transcriptional Responses in Zebrafish Larvae.

Bio Protoc 2018 Dec;8(23):e3100

MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe Road, Edinburgh, EH4 2XU, The United Kingdom.

Zebrafish have become an increasingly important model organism in the field of wound healing and regenerative medicine, due to their high regenerative capacity coupled with high-resolution imaging in living animals. In a recent study, we described multiple physical and chemical methods to induce notochord injury that led to highly specific transcriptional responses in notochord cellular subpopulations. The notochord is a critical embryonic structure that functions to shape and pattern the vertebrae and spinal column. Read More

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http://dx.doi.org/10.21769/BioProtoc.3100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6309551PMC
December 2018

Relative Quantitation of Polymerized Actin in Suspension Cells by Flow Cytometry.

Bio Protoc 2018 Nov;8(22)

Department of Biology, University of Massachusetts, Amherst MA, USA.

The amount of polymerized actin within a cell can vary widely due to natural processes and/or experimentally induced perturbations. We routinely use this protocol to measure relative polymerized actin content between cell populations by staining cells in suspension with fluorescent phalloidin, then measuring total cell fluorescence using flow cytometry. Although developed for human cells, we have easily adapted this method for use with diverse eukaryotic cell types. Read More

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http://dx.doi.org/10.21769/BioProtoc.3094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6301187PMC
November 2018

A Modified Barnes Maze for Juvenile Rats.

Bio Protoc 2018 Nov;8(22)

Krasnow Institute for Advanced Study, George Mason University, Fairfax, VA, USA.

To better understand neural codes for spatial navigation and to address cognitive development questions, the neurobiology of postnatal hippocampal development is receiving increased attention. While the Morris Water Maze is the gold standard for assessing hippocampal integrity, potential confounds can be difficult to control for in juvenile rodents (around three weeks of age, when spatial navigation ability first emerges) and this wet maze is not amenable to electrophysiological recording. A dry maze alternative with minimal training, like the Barnes Maze adapted for juvenile rats, can help overcome these obstacles. Read More

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http://dx.doi.org/10.21769/BioProtoc.3084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6294133PMC
November 2018

Quantification of Infectious Sendai Virus Using Plaque Assay.

Bio Protoc 2018 Nov;8(21)

Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, Los Angeles, USA.

Sendai virus (SeV) is an enveloped, single-stranded RNA virus of the family . SeV is a useful tool to study its infectious pathomechanism in immunology and the pathomechanism of a murine model of IgA nephropathy. Virus quantification is essential not only to determine the original viral titers for an appropriate application, but also to measure the viral titers in samples from the harvests from experiments. Read More

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https://bio-protocol.org/e3068
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http://dx.doi.org/10.21769/BioProtoc.3068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6289198PMC
November 2018
17 Reads

Microarray, IPA and GSEA Analysis in Mice Models.

Bio Protoc 2018 Sep;8(17)

Department of Animal Sciences, Purdue University, West Lafayette, United States.

This protocol details a method to analyze two tissue samples at the transcriptomic level using microarray analysis, ingenuity pathway analysis (IPA) and gene set enrichment analysis (GSEA). Methods such as these provide insight into the mechanisms underlying biological differences across two samples and thus can be applied to interrogate a variety of processes across different tissue samples, conditions, and the like. The full method detailed below can be applied to determine the effects of muscle-specific Notch1 activation in the mouse model and to analyze previously published microarray data of human liposarcoma cell lines. Read More

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http://dx.doi.org/10.21769/BioProtoc.2999DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6289195PMC
September 2018
1 Read

Generation of Stable Expression Mammalian Cell Lines Using Lentivirus.

Bio Protoc 2018 Nov;8(21)

Department of Radiation Oncology, Stanford University, Stanford, CA, USA.

Lentiviruses are used very widely to generate stable expression mammalian cell lines. They are used for both gene down-regulation (by using shRNA) or for gene up-regulation (by using ORF of gene of interest). The technique of generating stable cell lines using 3 generation lentivirus is very robust and it typically takes about 1-2 weeks to get stable expression for most mammalian cell lines. Read More

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https://bio-protocol.org/e3073
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http://dx.doi.org/10.21769/BioProtoc.3073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6261361PMC
November 2018
4 Reads

Eicosanoid Isolation from Mouse Intestinal Tissue for ELISA.

Authors:
Isabella Rauch

Bio Protoc 2018 Nov;8(21)

Division of Immunology & Pathogenesis, Department of Molecular & Cell Biology, University of California, Berkeley, CA 94720, USA.

Activation of inflammasomes in peritoneal macrophages and intestinal epithelial cells (IEC) leads to the release of eicosanoids. To assess the amount of eicosanoids released by IEC, lipids need to be isolated from whole tissue previous to analysis by lipid mass spectrometry or ELISA. This protocol describes how to isolate lipids from intestinal tissue for analysis by PGE-ELISA and normalize to tissue protein content. Read More

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http://dx.doi.org/10.21769/BioProtoc.3066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6258896PMC
November 2018
1 Read

Detection and Differentiation of Multiple Viral RNAs Using Branched DNA FISH Coupled to Confocal Microscopy and Flow Cytometry.

Bio Protoc 2018 Oct;8(20)

Department of Genetics, Stanford University School of Medicine, Stanford, CA, 94305, USA.

Due to the exceptionally high mutation rates of RNA-dependent RNA polymerases, infectious RNA viruses generate extensive sequence diversity, leading to some of the lowest barriers to the development of antiviral drug resistance in the microbial world. We have previously discovered that higher barriers to the development of drug resistance can be achieved through dominant suppression of drug-resistant viruses by their drug-susceptible parents. We have explored the existence of dominant drug targets in poliovirus, dengue virus and hepatitis C virus (HCV). Read More

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http://dx.doi.org/10.21769/BioProtoc.3058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6269108PMC
October 2018
1 Read

Transcytosis Assay for Transport of Glycosphingolipids across MDCK-II Cells.

Bio Protoc 2018 Oct;8(20)

Division of Gastroenterology, Boston Children's Hospital, Boston, Massachusetts 02115, USA.

Absorption and secretion of peptide and protein cargoes across single-cell thick mucosal and endothelial barriers occurs by active endocytic and vesicular trafficking that connects one side of the epithelial or endothelial cell (the lumen) with the other (the serosa or blood). Assays that assess this pathway must robustly control for non-specific and passive solute flux through weak or damaged intercellular junctions that seal the epithelial or endothelial cells together. Here we describe an cell culture Transwell assay for transcytosis of therapeutic peptides linked covalently to various species of the glycosphingolipid GM1. Read More

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http://dx.doi.org/10.21769/BioProtoc.3049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6261372PMC
October 2018
1 Read

Behavioral Evaluation of Seeking and Preference of Alcohol in Mice Subjected to Stress.

Bio Protoc 2018 Oct;8(20)

Department of Biology, William Paterson University of New Jersey, Wayne, USA.

The alcohol preference model is one of the most widely used animal models relevant to alcoholism. Stressors increase alcohol consumption. Here we present a protocol for a rapid and useful tool to test alcohol preference and stress-induced alcohol consumption in mice. Read More

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http://dx.doi.org/10.21769/BioProtoc.3061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6261307PMC
October 2018
4 Reads

A Method for SUMO Modification of Proteins .

Bio Protoc 2018 Oct;8(19)

Department of Biochemistry & Molecular Biology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA.

The Small Ubiquitin-related Modifier (SUMO) is a protein that is post-translationally added to and reversibly removed from other proteins in eukaryotic cells. SUMO and enzymes of the SUMO pathway are well conserved from yeast to humans and SUMO modification regulates a variety of essential cellular processes including transcription, chromatin remodeling, DNA damage repair, and cell cycle progression. One of the challenges in studying SUMO modification is the relatively low steady-state level of a SUMO-modified protein due in part to the activity of SUMO deconjugating enzymes known as SUMO Isopeptidases or SENPs. Read More

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https://bio-protocol.org/e3033
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http://dx.doi.org/10.21769/BioProtoc.3033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6261370PMC
October 2018
2 Reads

Culture and Lentiviral Transduction of Benign Prostatic Hyperplasia (BPH) Samples.

Bio Protoc 2018 11;8(21)

Newcastle Cancer Centre at the Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, England.

To assess oncogenic potential, classical transformation assays are based on cell line models. However, cell line based models do not reflect the complexity of human tissues. We thus developed an inducible expression system for gene expression in human tissues, which maintain native tissue architecture, such as epithelia and stroma. Read More

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6275124PMC
http://dx.doi.org/10.21769/BioProtoc.3075DOI Listing
November 2018
10 Reads

Kinetic Characterization of the Type Three Secretion System ATPase Spa47 Using α-P ATP.

Bio Protoc 2018 Nov;8(21)

Department of Chemistry and Biochemistry, Utah State University, Logan, USA.

ATPases represent a diverse class of enzymes that utilize ATP hydrolysis to support critical biological functions such as driving ion pumps, providing mechanical work, unfolding/folding proteins, and supporting otherwise thermodynamically unfavorable chemical reactions. We have recently shown that the protein Spa47 is an ATPase that supports protein secretion through its specialized type three secretion apparatus (T3SA), supporting infection of human host cells. Characterizing ATPases, such as Spa47, requires a means to accurately determine enzyme activity (ATP hydrolysis) as a function of time, reaction conditions, and potential cofactors, regulators, inhibitors, . Read More

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https://bio-protocol.org/e3074
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http://dx.doi.org/10.21769/BioProtoc.3074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6247913PMC
November 2018
7 Reads

Electroporation of Labeled Antibodies to Visualize Endogenous Proteins and Posttranslational Modifications in Living Metazoan Cell Types.

Bio Protoc 2018 Nov;8(21)

Université de Strasbourg, 67404, Illkirch, France.

The spatiotemporal localization of different intracellular factors in real-time and their detection in live cells are important parameters to understand dynamic protein-based processes. Therefore, there is a demand to perform live-cell imaging and to measure endogenous protein dynamics in single cells. However, fluorescent labeling of endogenous protein in living cells without overexpression of fusion proteins or genetic tagging has not been routinely possible. Read More

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http://dx.doi.org/10.21769/BioProtoc.3069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6245562PMC
November 2018
1 Read

Nuclear/Cytoplasmic Fractionation of Proteins from .

Bio Protoc 2018 Oct;8(20)

European Research Institute for the Biology of Ageing, University of Groningen, University Medical Centre Groningen, Groningen, the Netherlands.

is widely used to investigate biological processes related to health and disease. To study protein localization, fluorescently-tagged proteins can be used or immunohistochemistry can be performed in whole worms. Here, we describe a technique to localize a protein of interest at a subcellular level in lysates, which can give insight into the location, function and/or toxicity of proteins. Read More

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http://dx.doi.org/10.21769/BioProtoc.3053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6245569PMC
October 2018
15 Reads

Filter Retardation Assay for Detecting and Quantifying Polyglutamine Aggregates Using Lysates.

Bio Protoc 2018 Oct;8(19)

European Research Institute for the Biology of Ageing, University of Groningen, University Medical Centre Groningen, Groningen, the Netherlands.

Protein aggregation is a hallmark of several neurodegenerative diseases and is associated with impaired protein homeostasis. This imbalance is caused by the loss of the protein's native conformation, which ultimately results in its aggregation or abnormal localization within the cell. Using a model of polyglutamine diseases, we describe in detail the filter retardation assay, a method that captures protein aggregates in a cellulose acetate membrane and allows its detection and quantification by immunoblotting. Read More

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https://bio-protocol.org/e3042
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http://dx.doi.org/10.21769/BioProtoc.3042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6235101PMC
October 2018
12 Reads

Fluorescence Titrations to Determine the Binding Affinity of Cyclic Nucleotides to SthK Ion Channels.

Bio Protoc 2018 Oct;8(19)

Weill Cornell Medicine, Department of Anesthesiology, New York, NY, USA.

The cyclic-nucleotide modulated ion channel family includes cyclic nucleotide-gated (CNG) and hyperpolarization-activated and cyclic nucleotide-modulated (HCN) channels, which play essential roles in visual and olfactory signaling and the heart pacemaking activity. Functionally, these channels have been extensively characterized by electrophysiological techniques from protein heterologously expressed in Xenopus oocytes and mammalian cells. On the other hand, expression and purification of these proteins for biophysical and structural analyses is problematic and expensive and, accordingly, only limited information on the purified channels is available in the literature. Read More

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https://bio-protocol.org/e3041
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http://dx.doi.org/10.21769/BioProtoc.3041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6221198PMC
October 2018
17 Reads

Artificial Inhalation Protocol in Adult Mice.

Bio Protoc 2018 Sep;8(18)

Department of Neurobiology and Anatomy, University of Utah, Salt Lake City, UT, USA.

Research in the area of olfactory physiology benefits from having direct access to the nasal airways through which odorants can be presented. Ordinarily, the passage of odorants through the airways is controlled by respiratory rhythm. This fact makes it difficult to control the timing and strength of an olfactory stimulus, since animals must breathe regularly, and the act of breathing itself also controls odorant presentation. Read More

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http://dx.doi.org/10.21769/BioProtoc.3024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6221193PMC
September 2018

Mammalian Cell-derived Vesicles for the Isolation of Organelle Specific Transmembrane Proteins to Conduct Single Molecule Studies.

Bio Protoc 2018 May;8(9)

Department of Chemistry, University of Kentucky, Lexington, Kentucky, USA.

Cell-derived vesicles facilitate the isolation of transmembrane proteins in their physiological membrane maintaining their structural and functional integrity. These vesicles can be generated from different cellular organelles producing, housing, or transporting the proteins. Combined with single-molecule imaging, isolated organelle specific vesicles can be employed to study the trafficking and assembly of the embedded proteins. Read More

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https://bio-protocol.org/e2825
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http://dx.doi.org/10.21769/BioProtoc.2825DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6217831PMC
May 2018
4 Reads

Testing for Assortative Mating by Diet in .

Bio Protoc 2018 Oct;8(20)

School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK.

Experimental studies of the evolution of reproductive isolation in real time are a powerful way to reveal the way that fundamental processes, such as mate choice, initiate divergence. Mate choice, while frequently described in females, can occur in either sex, and can be affected by the genetics or environment of an individual. Here we describe simple protocols for assessing mating outcomes in fruit flies, which in this context can be used to assess reproductive isolation derived from rearing on different diets over multiple generations. Read More

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http://dx.doi.org/10.21769/BioProtoc.3057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6217913PMC
October 2018

An Image Analysis Pipeline to Quantify Emerging Cracks in Materials or Adhesion Defects in Living Tissues.

Bio Protoc 2018 Oct;8(19):e3036

RDP, Université de Lyon, ENS de Lyon, UCB Lyon 1, CNRS, INRA, INRIA, Lyon, France.

Microcracks in materials reflect their mechanical properties. The quantification of the number or orientation of such cracks is thus essential in many fields, including engineering and geology. In biology, cracks in soft tissues can reflect adhesion defects, and the analysis of their pattern can help to deduce the magnitude and orientation of tensions in organs and tissues. Read More

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https://bio-protocol.org/e3036
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http://dx.doi.org/10.21769/BioProtoc.3036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6218008PMC
October 2018
13 Reads

6-hydroxydopamine (6-OHDA) Oxidative Stress Assay for Observing Dopaminergic Neuron Loss in .

Bio Protoc 2018 Sep;8(18)

Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, United Kingdom.

The nematode is a powerful genetic model that can be used to investigate neuronal death. Research using has been crucial to characterize cell death programmes that are conserved in mammals. Many neuronal signaling components, such as those mediating dopaminergic neurotransmission, are preserved as well. Read More

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http://dx.doi.org/10.21769/BioProtoc.3025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6218003PMC
September 2018
1 Read

Sendai Virus Propagation Using Chicken Eggs.

Bio Protoc 2018 Sep;8(18)

Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, Los Angeles, USA.

Sendai virus is a member of the family , and an enveloped virus with a negative-stranded RNA genome. Sendai virus is not pathogenic to humans, but for mice and can cause pneumonia in mice. Easy and efficient techniques for propagating Sendai virus are required for studying virus replication, virus-induced innate- and adaptive-immunity, Sendai-virus-based virotherapy and IgA nephropathy. Read More

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https://bio-protocol.org/e3009
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http://dx.doi.org/10.21769/BioProtoc.3009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6200407PMC
September 2018
2 Reads

Microfluidics-Based Analysis of Contact-dependent Bacterial Interactions.

Bio Protoc 2018 Aug;8(16)

BioCircuits Institute, University of California, San Diego, La Jolla, CA, United States.

Bacteria in nature live in complex communities with multiple cell types and spatially-dependent interactions. Studying cells in well-mixed environments such as shaking culture tubes or flasks cannot capture these spatial dynamics, but cells growing in full-fledged biofilms are difficult to observe in real time. We present here a protocol for observing time-resolved, multi-species interactions at single-cell resolution. Read More

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https://bio-protocol.org/e2970
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http://dx.doi.org/10.21769/BioProtoc.2970DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6200413PMC
August 2018
38 Reads

Deoxycholate Fractionation of Fibronectin (FN) and Biotinylation Assay to Measure Recycled FN Fibrils in Epithelial Cells.

Bio Protoc 2018 Aug;8(16)

Department of Chemistry and Biochemistry, University of South Carolina, Columbia, USA.

Fibronectin (FN) is an extracellular matrix protein that is secreted by many cell types and binds predominantly to the cell surface receptor Integrin α5β1. Integrin α5β1 binding initiates the step-wise assembly of FN into fibrils, a process called fibrillogenesis. We and several others have demonstrated critical effects of fibrillogenesis on cell migration and metastasis. Read More

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https://bio-protocol.org/e2972
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http://dx.doi.org/10.21769/BioProtoc.2972DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6200405PMC
August 2018
18 Reads

Preparation of Sequencing RNA Libraries through Chemical Cross-linking Coupled to Affinity Purification (cCLAP) in .

Bio Protoc 2018 Oct;8(19):e3029

Growth & Development, Biozentrum, University of Basel, Basel, Switzerland.

Ribonucleoprotein particles (mRNPs) are complexes consisting of mRNAs and RNA-binding proteins (RBPs) which control mRNA transcription localization, turnover, and translation. Some mRNAs within the mRNPs have been shown to undergo degradation or storage. Those transcripts can lack general mRNA elements, like the poly(A) tail or 5' cap structure, which prevent their identification through the application of widely-used approaches like oligo(dT) purification. Read More

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http://dx.doi.org/10.21769/BioProtoc.3029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6192512PMC
October 2018
2 Reads

High-throughput Microscopic Analysis of Invasion of Host Cells.

Bio Protoc 2018 Sep;8(18)

Dynamics of Host-Pathogen Interactions Unit, Institut Pasteur, Paris, France.

is a Gram-negative bacterium causing a gastro-enteric disease called salmonellosis. During the first phase of infection, uses its flagella to swim near the surface of the epithelial cells and to target specific site of infection. In order to study the selection criteria that determine which host cells are targeted by the pathogen, and to analyze the relation between infecting (, cooperation or competition), we have established a high-throughput microscopic assay of HeLa cells sequentially infected with fluorescent bacteria. Read More

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https://bio-protocol.org/e3017
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http://dx.doi.org/10.21769/BioProtoc.3017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195197PMC
September 2018
10 Reads