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Bio-protocol

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https://www.readbyqxmd.com/read/29021994/stereotaxic-adeno-associated-virus-injection-and-cannula-implantation-in-the-dorsal-raphe-nucleus-of-mice
#1
Patrícia A Correia, Sara Matias, Zachary F Mainen
Optogenetic methods are now widespread in neuroscience research. Here we present a detailed surgical procedure to inject adeno-associated viruses and implant optic fiber cannulas in the dorsal raphe nucleus (DRN) of living mice. Combined with transgenic mouse lines, this protocol allows specific targeting of serotonin-producing neurons in the brain. It includes fixing a mouse in a stereotaxic frame, performing a craniotomy, virus injection and fiber implantation. Animals can be later used in behavioral experiments, combined with optogenetic manipulations (Dugué et al...
September 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28979923/isolation-of-mouse-cardiac-neural-crest-cells-and-their-differentiation-into-smooth-muscle-cells
#2
Xia Wang, Sophie Astrof
Cardiac neural crest cells (CNCCs) originate at the dorsal edge of the neural tube between the otic pit and the caudal edge of the 3(rd) somite, and migrate into the pharyngeal arches and the heart. We have shown that fibronectin (Fn1) plays an important role in the development of the CNCC by regulating the differentiation of CNCCs into vascular smooth muscle cells around pharyngeal arch arteries (Wang and Astrof, 2016). This protocol describes the isolation of CNCCs from the neural tube and from the caudal pharyngeal arches, and the differentiation of neural crest-derived cells into smooth muscle cells...
September 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28966949/protocol-for-establishing-a-multiplex-image-based-autophagy-rnai-screen-in-cell-cultures
#3
Jennifer Jung, Christian Behrends
Autophagy is a recycling pathway, in which intracellular cargoes including protein aggregates and bacteria are engulfed by autophagosomes and subsequently degraded after fusion with lysosomes. Dysregulation of this process is involved in several human diseases such as cancer or neurodegeneration. Hence, advancing our understanding of how autophagy is regulated provides an opportunity to explore druggable targets and subsequently develop treatment strategies for these diseases. To identify novel autophagy regulators, we developed an image-based phenotypic RNAi screening approach using autophagic marker proteins at endogenous levels (Jung et al...
September 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28966948/labelling-halotag-fusion-proteins-with-halotag-ligand-in-living-cells
#4
Huy Nguyen Duc, Xiaojun Ren
HaloTag has been widely used to label proteins in vitro and in vivo (Los et al., 2008). In this protocol, we describe labelling HaloTag-Cbx fusion proteins by HaloTag ligands for live-cell single-molecule imaging (Zhen et al., 2016).
September 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28966947/purifying-properly-folded-cysteine-rich-zinc-finger-containing-recombinant-proteins-for-structural-drug-targeting-studies-the-ch1-domain-of-p300-as-a-case-example
#5
Yong Joon Kim, Stefan Kaluz, Anil Mehta, Emily Weinert, Shannon Rivera, Erwin G Van Meir
The transcription factor Hypoxia-Inducible Factor (HIF) complexes with the coactivator p300, activating the hypoxia response pathway and allowing tumors to grow. The CH1 and CAD domains of each respective protein form the interface between p300 and HIF. Small molecule compounds are in development that target and inhibit HIF/p300 complex formation, with the goal of reducing tumor growth. High resolution NMR spectroscopy is necessary to study ligand interaction with p300-CH1, and purifying high quantities of properly folded p300-CH1 is needed for pursuing structural and biophysical studies...
September 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28966946/expression-and-purification-of-a-mammalian-p2x7-receptor-from-sf9-insect-cells
#6
Akira Karasawa, Toshimitsu Kawate
The P2X7 receptor is an extracellular ATP-gated ion channel found only in eukaryotes (Bartlett et al., 2014). Due to its unique properties among P2X receptors, such as formation of a large conductance pore, the P2X7 receptor has been implicated in devastating diseases like chronic pain (North and Jarvis, 2013). However, mechanisms underlying the P2X7 specific properties remain poorly understood, partly because purification of this eukaryotic membrane protein has been challenging. Here we describe a detailed protocol for expressing and purifying a mammalian P2X7 receptor using an insect cell-baculovirus system...
September 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28936468/gfp-grb2-translocation-assay-using-high-content-imaging-to-screen-for-modulators-of-egfr-signaling
#7
Julia Petschnigg, Robin Ketteler
High-content screening is a useful tool to understand complex cellular processes and to identify genes, proteins or small molecule compounds that modulate such pathways. High-content assays monitor the function of a protein or pathway by visualizing a change in an image-based readout, such as a change in the localization of a reporter protein. Examples of this can be the translocation of a fluorescently tagged protein from the cytoplasm to the nucleus or to the plasma membrane. One protein that is known to undergo such translocation is the Growth Factor Receptor-bound protein 2 (GRB2) that is recruited to the plasma membrane upon stimulation of a growth factor receptor and subsequently undergoes internalization...
September 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28936467/peroxisome-motility-measurement-and-quantification-assay
#8
Jeremy Metz, Inês G Castro, Michael Schrader
Organelle movement, distribution and interaction contribute to the organisation of the eukaryotic cell. Peroxisomes are multifunctional organelles which contribute to cellular lipid metabolism and ROS homeostasis. They distribute uniformly in mammalian cells and move along microtubules via kinesin and dynein motors. Their metabolic cooperation with mitochondria and the endoplasmic reticulum (ER) is essential for the β-oxidation of fatty acids and the synthesis of myelin lipids and polyunsaturated fatty acids...
September 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28966945/a-high-throughput-assay-for-mrna-silencing-in-primary-cortical-neurons-in-vitro-with-oligonucleotide-therapeutics
#9
Julia F Alterman, Andrew H Coles, Lauren M Hall, Neil Aronin, Anastasia Khvorova, Marie-Cécile Didiot
Primary neurons represent an ideal cellular system for the identification of therapeutic oligonucleotides for the treatment of neurodegenerative diseases. However, due to the sensitive nature of primary cells, the transfection of small interfering RNAs (siRNA) using classical methods is laborious and often shows low efficiency. Recent progress in oligonucleotide chemistry has enabled the development of stabilized and hydrophobically modified small interfering RNAs (hsiRNAs). This new class of oligonucleotide therapeutics shows extremely efficient self-delivery properties and supports potent and durable effects in vitro and in vivo...
August 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28932764/antisense-oligonucleotide-mediated-knockdown-in-mammary-tumor-organoids
#10
Sarah D Diermeier, David L Spector
Primary mammary tumor organoids grown in 3D are an excellent system to study tumor biology. They resemble the organization and physiology of native epithelia more closely than cancer cell lines grown in 2D, and additionally model interactions with the ECM (Boj et al., 2015; Clevers, 2016; Shamir and Ewald, 2014). Mammary tumor organoids are therefore a promising model system to identify and characterize novel drivers of breast cancer that would be unlikely to be identified using 2D cell lines. Antisense oligonucleotides can be used to efficiently and specifically knockdown target genes in the cell (Bennett et al...
August 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28932763/a-co-culture-assay-to-determine-efficacy-of-tnf-%C3%AE-suppression-by-biomechanically-induced-human-bone-marrow-mesenchymal-stem-cells
#11
Miguel F Diaz, Siobahn M Evans, Scott D Olson, Charles S Cox, Pamela L Wenzel
The beneficial effects of mesenchymal stem cell (MSC)-based cellular therapies are believed to be mediated primarily by the ability odansf MSCs to suppress inflammation associated with chronic or acute injury, infection, autoimmunity, and graft-versus-host disease. To specifically address the effects of frictional force caused by blood flow, or wall shear stress (WSS), on human MSC immunomodulatory function, we have utilized microfluidics to model WSS at the luminal wall of arteries. Anti-inflammatory potency of MSCs was subsequently quantified via measurement of TNF-α production by activated murine splenocytes in co-culture assays...
August 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28920069/assessment-of-thermal-pain-sensation-in-rats-and-mice-using-the-hargreaves-test
#12
Menghon Cheah, James W Fawcett, Melissa R Andrews
The Hargreaves test is specifically designed to assess thermal pain sensation in rodents such as rats and mice. This test has been used in experiments involving pain sensitization or recovery of thermal pain response following neural injury and regeneration. We present here a step-by-step protocol highlighted with important notes to guide first-time users through the learning process. Additionally, we have also included representative data from a rat model of sensory denervation showing how the data can be analysed to obtain meaningful results...
August 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28868333/rapid-profiling-cell-cycle-by-flow-cytometry-using-concurrent-staining-of-dna-and-mitotic-markers
#13
Yuqing Shen, Paolo Vignali, Ruoning Wang
The flow cytometric quantitation of DNA content by DNA-binding fluorochrome, propidium iodide (PI) is the most widely used method for cell cycle analysis. However, the commonly used methods are time-consuming and labor-intensive and are incompatible with staining of mitotic markers by fluorescent-labeled antibodies. Here, we report an optimized simple protocol for rapid and simultaneous analysis of characteristic mitotic phosphorylated proteins and DNA content, permitting the quantification of cells in mitosis, G1, S and G2 stage in a single assay...
August 20, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28936466/mouse-m%C3%A3-ller-cell-isolation-and-culture
#14
Xiao Liu, Luosheng Tang, Yongqing Liu
Müller cells are the major supportive and protective glial cells across the retina. Unlike in fish, they have lost the capacity to regenerate the retina in mammals. But, mammalian Müller cells still retain certain retinal stem cell properties with various degree of self-renewal and differentiation potentials, and thereby held a merit in cell-based therapies for treating retinal degeneration diseases. In our laboratory, we use an enzymatic procedure to isolate, purify, and culture mouse Müller cells.
August 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28932762/liposome-disruption-assay-to-examine-lytic-properties-of-biomolecules
#15
John R Jimah, Paul H Schlesinger, Niraj H Tolia
Proteins may have three dimensional structural or amino acid features that suggest a role in targeting and disrupting lipids within cell membranes. It is often necessary to experimentally investigate if these proteins and biomolecules are able to disrupt membranes in order to conclusively characterize the function of these biomolecules. Here, we describe an in vitro assay to evaluate the membrane lytic properties of proteins and biomolecules. Large unilamellar vesicles (liposomes) containing carboxyfluorescein at fluorescence-quenching concentrations are treated with the biomolecule of interest...
August 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28932761/digestion-of-peptidoglycan-and-analysis-of-soluble-fragments
#16
Ryan E Schaub, Joseph P Dillard
Peptidoglycan (murein) is a vital component of the cell wall of nearly all bacteria, composed of sugars linked by short peptides. This protocol describes the purification of macromolecular peptidoglycan from cultured bacteria and the analysis of enzyme-digested peptidoglycan fragments using high performance liquid chromatography (HPLC). Digested peptidoglycan fragments can be identified by mass spectrometry, or predicted by comparing retention times with other published chromatograms. The quantitative nature of this method allows for the measurement of changes to peptidoglycan composition between different species of bacteria, growth conditions, or mutations...
August 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28932760/membrane-lipid-screen-to-identify-molecular-targets-of-biomolecules
#17
John R Jimah, Paul H Schlesinger, Niraj H Tolia
Proteins that bind to and disrupt cell membranes may target specific phospholipids. Here we describe a protocol to identify the lipid targets of proteins and biomolecules. First, we describe a screen to identify lipids in membranes that are specifically bound by the biomolecule of interest. Second, we describe a method for determining if the presence of these lipids within membranes is necessary for membrane disruption. The methods described here were used to determine that the malaria vaccine candidate CelTOS disrupts cell membranes by specifically targeting phosphatidic acid (Jimah et al...
August 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28868332/primary-culture-system-for-germ-cells-from-caenorhabditis-elegans-tumorous-germline-mutants
#18
Alexandra S Vagasi, Mohammad M Rahman, Snehal N Chaudhari, Edward T Kipreos
The Caenorhabditis elegans germ line is an important model system for the study of germ stem cells. Wild-type C. elegans germ cells are syncytial and therefore cannot be isolated in in vitro cultures. In contrast, the germ cells from tumorous mutants can be fully cellularized and isolated intact from the mutant animals. Here we describe a detailed protocol for the isolation of germ cells from tumorous mutants that allows the germ cells to be maintained for extended periods in an in vitro primary culture. This protocol has been adapted from Chaudhari et al...
August 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28804739/selection-of-genetically-modified-bacteriophages-using-the-crispr-cas-system
#19
Miriam Manor, Udi Qimron
We present a CRISPR-Cas based technique for deleting genes from the T7 bacteriophage genome. A DNA fragment encoding homologous arms to the target gene to be deleted is first cloned into a plasmid. The T7 phage is then propagated in Escherichia coli harboring this plasmid. During this propagation, some phage genomes undergo homologous recombination with the plasmid, thus deleting the targeted gene. To select for these genomes, the CRISPR-Cas system is used to cleave non-edited genomes, enabling isolation of the desired recombinant phages...
August 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28904991/bioelectrospray-methodology-for-dissection-of-the-host-pathogen-interaction-in-human-tuberculosis
#20
Liku B Tezera, Magdalena K Bielecka, Paul T Elkington
Standard cell culture models have been used to investigate disease pathology and to test new therapies for over fifty years. However, these model systems have often failed to mimic the changes occurring within three-dimensional (3-D) space where pathology occurs in vivo. To truthfully represent this, an emerging paradigm in biology is the importance of modelling disease in a physiologically relevant 3-D environment. One of the approaches for 3-D cell culture is bioelectrospray technology. This technique uses an alginate-based 3-D environment as an inert backbone within which mammalian cells and extracellular matrix can be incorporated...
July 20, 2017: Bio-protocol
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