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Bio-protocol

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https://www.readbyqxmd.com/read/28018942/murine-leukemia-virus-mlv-based-coronavirus-spike-pseudotyped-particle-production-and-infection
#1
Jean Kaoru Millet, Gary R Whittaker
Viral pseudotyped particles (pp) are enveloped virus particles, typically derived from retroviruses or rhabdoviruses, that harbor heterologous envelope glycoproteins on their surface and a genome lacking essential genes. These synthetic viral particles are safer surrogates of native viruses and acquire the tropism and host entry pathway characteristics governed by the heterologous envelope glycoprotein used. They have proven to be very useful tools used in research with many applications, such as enabling the study of entry pathways of enveloped viruses and to generate effective gene-delivery vectors...
December 5, 2016: Bio-protocol
https://www.readbyqxmd.com/read/27928550/ionization-properties-of-phospholipids-determined-by-zeta-potential-measurements
#2
Murugappan Sathappa, Nathan N Alder
Biological membranes are vital for diverse cellular functions such as maintaining cell and organelle structure, selective permeability, active transport, and signaling. The surface charge of the membrane bilayer plays a critical role in these myriad processes. For most biomembranes, the surface charge of anionic phospholipids contributes to the negative surface charge density within the interfacial region of the bilayer. To quantify surface charge, it is essential to understand the proton dissociation behavior of the titratable headgroups within such lipids...
November 20, 2016: Bio-protocol
https://www.readbyqxmd.com/read/27840835/generation-of-mitochondrial-nuclear-exchange-mice-via-pronuclear-transfer
#3
Robert A Kesterson, Larry W Johnson, Laura J Lambert, Jay L Vivian, Danny R Welch, Scott W Ballinger
The mitochondrial paradigm for common disease proposes that mitochondrial DNA (mtDNA) sequence variation can contribute to disease susceptibility and progression. To test this concept, we developed the Mitochondrial-nuclear eXchange (MNX) model, in which isolated embryonic pronuclei from one strain of species are implanted into an enucleated embryo of a different strain of the same species (e.g., C57BL/6 and C3H/HeN, Mus musculus), generating a re-constructed zygote harboring nuclear and mitochondrial genomes from different strains...
October 20, 2016: Bio-protocol
https://www.readbyqxmd.com/read/27785458/aniline-blue-and-calcofluor-white-staining-of-callose-and-cellulose-in-the-streptophyte-green-algae-zygnema-and-klebsormidium
#4
Klaus Herburger, Andreas Holzinger
Plant including green algal cells are surrounded by a cell wall, which is a diverse composite of complex polysaccharides and crucial for their function and survival. Here we describe two simple protocols to visualize callose (β-1→3-glucan) and cellulose (β-1→4-glucan) and related polysaccharides in the cell walls of streptophyte green algae by using standard dyes and epifluorescence microscopy.
October 20, 2016: Bio-protocol
https://www.readbyqxmd.com/read/27819014/isolation-culture-and-staining-of-single-myofibers
#5
Yann Simon Gallot, Sajedah M Hindi, Aman K Mann, Ashok Kumar
Adult skeletal muscle regeneration is orchestrated by a specialized population of adult stem cells called satellite cells, which are localized between the basal lamina and the plasma membrane of myofibers. The process of satellite cell-activation, proliferation, and subsequent differentiation that occurs during muscle regeneration can be recapitulated ex vivo by isolation of single myofibers from skeletal muscles and culturing them under suspension conditions. Here, we describe an improved protocol to evaluate ex vivo satellite cells activation through isolation of single myofibers from extensor digitorum longus (EDL) muscle of mice and culturing and staining of myofiber-associated satellite cells with the markers of self-renewal, proliferation, and differentiation...
October 5, 2016: Bio-protocol
https://www.readbyqxmd.com/read/27642615/analysis-of-enteric-neural-crest-cell-migration-using-heterotopic-grafts-of-embryonic-guts
#6
Rodolphe Soret, Nicolas Pilon
Hirschsprung disease (HSCR), also named aganglionic megacolon, is a severe congenital malformation characterized by a lack of enteric nervous system (ENS) in the terminal regions of the bowel (Bergeron et al., 2013). As the ENS notably regulates motility in the whole gastrointestinal track, the segment without neurons remains tonically contracted, resulting in functional intestinal obstruction and accumulation of fecal material (megacolon). HSCR occurs when enteric neural progenitors of vagal neural crest origin fail to fully colonize the developing intestines...
September 5, 2016: Bio-protocol
https://www.readbyqxmd.com/read/28093578/mouse-liver-mitochondria-isolation-size-fractionation-and-real-time-momp-measurement
#7
Thibaud T Renault, Mark P A Luna-Vargas, Jerry E Chipuk
The mitochondrial pathway of apoptosis involves a complex interplay between dozens of proteins and lipids, and is also dependent on the shape and size of mitochondria. The use of cellular models in past studies has not been ideal for investigating how the complex multi-factor interplay regulates the molecular mechanisms of mitochondrial outer membrane permeabilization (MOMP). Isolated systems have proven to be a paradigm to deconstruct MOMP into individual steps and to study the behavior of each subset of MOMP regulators...
August 5, 2016: Bio-protocol
https://www.readbyqxmd.com/read/28042595/expression-purification-and-crystallization-of-the-herpesvirus-nuclear-egress-complex-nec
#8
Janna M Bigalke, Ekaterina E Heldwein
The protocol describes the production and crystallization of the soluble form of the nuclear egress complex (NEC) from Herpes simplex virus 1 and Pseudorabies virus. The NEC is a heterodimer that consists of conserved proteins UL31 and UL34. NEC oligomerization deforms the inner nuclear membrane around the capsid in infected cells, thereby mediating capsid budding into the perinuclear space during nuclear egress. We have successfully developed a protocol for large-scale preparation of highly pure NEC from two different viruses in a prokaryotic expression system, which enabled us to crystallize these viral protein complexes and determine their structures...
July 20, 2016: Bio-protocol
https://www.readbyqxmd.com/read/27366759/aorta-atherosclerosis-lesion-analysis-in-hyperlipidemic-mice
#9
Sarajo Mohanta, Changjun Yin, Christian Weber, Desheng Hu, Andreas Jr Habenicht
Atherosclerosis is a chronic inflammatory disease of large and medium-sized arteries. Apolipoprotein E-deficient (ApoE(-/-)) mice are used as experimental models to study human atherosclerosis. ApoE(-/-) mice are constitutively hyperlipidemic and develop intima plaques that resemble human plaques. Various issues including experimental design for lesion analysis, dietary conditions, isolation of the aorta, staining methods, morphometry, group size, age, the location within the arterial tree, and statistical analyses are important parameters that need to be addressed to obtain robust data...
June 5, 2016: Bio-protocol
https://www.readbyqxmd.com/read/27335895/preparation-of-single-cell-suspensions-from-mouse-aorta
#10
Desheng Hu, Changjun Yin, Sarajo K Mohanta, Christian Weber, Andreas J R Habenicht
Atherosclerosis is a chronic inflammatory disease of the arterial wall characterized by lipid deposition, plaque formation, and immune cell infiltration. Innate and adaptive immune cells infiltrate the artery during development of the disease. Moreover, advanced disease leads to formation of artery tertiary lymphoid organs in the adventitia (Grabner et al., 2009; Hu et al., 2015). Various and diverse types of immune cells have been identified in the aorta adventitia vs atherosclerotic plaques (Elewa et al., 2016; Galkina et al...
June 5, 2016: Bio-protocol
https://www.readbyqxmd.com/read/27231711/visualization-of-intracellular-tyrosinase-activity-in-vitro
#11
Riddhi Atul Jani, Sudeshna Nag, Subba Rao Gangi Setty
Melanocytes produce the melanin pigments in melanosomes and these organelles protect the skin against harmful ultraviolet rays. Tyrosinase is the key cuproenzyme which initiates the pigment synthesis using its substrate amino acid tyrosine or L-DOPA (L-3, 4-dihydroxyphenylalanine). Moreover, the activity of tyrosinase directly correlates to the cellular pigmentation. Defects in tyrosinase transport to melanosomes or mutations in the enzyme or reduced intracellular copper levels results in loss of tyrosinase activity in melanosomes, commonly observed in albinism...
April 20, 2016: Bio-protocol
https://www.readbyqxmd.com/read/27559541/methods-for-detecting-microbial-methane-production-and-consumption-by-gas-chromatography
#12
Jared T Aldridge, Jennie L Catlett, Megan L Smith, Nicole R Buan
Methane is an energy-dense fuel but is also a greenhouse gas 25 times more detrimental to the environment than CO2. Methane can be produced abiotically by serpentinization, chemically by Sabatier or Fisher-Tropsh chemistry, or biotically by microbes (Berndt et al., 1996; Horita and Berndt, 1999; Dry, 2002; Wolfe, 1982; Thauer, 1998; Metcalf et al., 2002). Methanogens are anaerobic archaea that grow by producing methane gas as a metabolic byproduct (Wolfe, 1982; Thauer, 1998). Our lab has developed and optimized three different gas chromatograph-utilizing assays to characterize methanogen metabolism (Catlett et al...
April 5, 2016: Bio-protocol
https://www.readbyqxmd.com/read/27468425/measurement-of-inositol-triphosphate-levels-from-rat-hippocampal-slices
#13
Nino Tabatadze, Catherine Woolley
Inositol triphosphate (IP3) is an important second messenger that participates in signal transduction pathways in diverse cell types including hippocampal neurons. Stimulation of phospholipase C in response to various stimuli (hormones, growth factors, neurotransmitters, neurotrophins, neuromodulators, odorants, light, etc) results in hydrolysis of phosphatidylinositol 4, 5-bisphosphate (PIP2), a phospholipid that is located in the plasma membrane, and leads to the production of IP3 and diacylglycerol. Binding of IP3 to the IP3 receptor (IP3R) induces Ca(2+) release from intracellular stores and enables the initiation of intracellular Ca(2+)-dependent signaling...
April 5, 2016: Bio-protocol
https://www.readbyqxmd.com/read/27213172/extraction-and-quantification-of-tryptophan-and-kynurenine-from-cultured-cells-and-media-using-a-high-performance-liquid-chromatography-hplc-system-equipped-with-an-ultra-sensitive-diode-array-detector
#14
Jeffrey Kim, Benjamin Stewart, Robert H Weiss
Evidence of the involvement of tryptophan and its metabolite, kynurenine, in various biological processes including cancer is constantly expanding. Analysis of cell extracts and culture media can allow for quick snapshots of the metabolic fluctuations occurring in vitro. Here, we describe a method for metabolite extraction from mammalian cells and analysis of extracted metabolites and cell culture media by HPLC with detection using an ultra-sensitive diode array detector.
April 5, 2016: Bio-protocol
https://www.readbyqxmd.com/read/27239483/in-vitro-differentiation-of-murine-innate-lymphoid-cells-from-common-lymphoid-progenitor-cells
#15
Corey Seehus, Jonathan Kaye
Subtypes of innate lymphoid cells (ILC), defined based on their cytokine secretion profiles and transcription factor expression, are important for host protection from pathogens and maintaining tissue homeostasis. ILCs develop from common lymphoid progenitors (CLP) in the bone marrow. Using the methods described here, we have previously shown that loss of the transcriptional regulator TOX (Thymocyte-selection associated HMG-box protein) leads to specific changes in ILC development and differentiation. Here, we describe how to obtain ILCs from in vivo isolated CLP grown in vitro...
March 20, 2016: Bio-protocol
https://www.readbyqxmd.com/read/27446983/mouse-mammary-intraductal-mind-method-for-transplantation-of-patient-derived-primary-dcis-cells-and-cell-lines
#16
Frances Kittrell, Kelli Valdez, Hanan Elsarraj, Yan Hong, Daniel Medina, Fariba Behbod
The MIND method involves intraductal injection of patient derived ductal carcinoma in situ (DCIS) cells and DCIS cell lines (MCF10DCIS.COM and SUM225) inside the mouse mammary ducts [Video 1 and Figure 1 in Behbod et al. (2009)]. This method mimics the normal environment of DCIS and facilitates study of the natural progression of human DCIS, i.e., their initial growth as carcinoma in situ within the ducts, followed by invasion into the stroma through the myoepithelial cell layer and basement membrane (Behbod et al...
March 5, 2016: Bio-protocol
https://www.readbyqxmd.com/read/27570799/isolation-culture-and-maintenance-of-mouse-intestinal-stem-cells
#17
Kevin P O'Rourke, Sarah Ackerman, Lukas E Dow, Scott W Lowe
In this protocol we describe our modifications to a method to isolate, culture and maintain mouse intestinal stem cells as crypt-villus forming organoids. These cells, isolated either from the small or large intestine, maintain self-renewal and multilineage differentiation potential over time. This provides investigators a tool to culture wild type or transformed intestinal epithelium, and a robust assay for stem cell tissue homeostasis in vitro.
February 20, 2016: Bio-protocol
https://www.readbyqxmd.com/read/27570798/immunofluorescent-staining-of-mouse-intestinal-stem-cells
#18
Kevin P O'Rourke, Lukas E Dow, Scott W Lowe
Immunofluorescent staining of organoids can be performed to visualize molecular markers of cell behavior. For example, cell proliferation marked by incorporation of nucleotide (EdU), or to observe markers of intestinal differentiation including paneth cells, goblet cells, or enterocytes (see Figure 1). In this protocol we detail a method to fix, permeabilize, stain and mount intestinal organoids for analysis by immunofluorescent confocal microscopy.
February 20, 2016: Bio-protocol
https://www.readbyqxmd.com/read/27446982/quantification-of-ethylene-production-in-tomato-leaves-infected-by-xanthomonas-euvesicatoria
#19
Jung-Gun Kim, William Stork, Mary Beth Mudgett
Ethylene is a gaseous plant hormone controlling fruit ripening, flower opening, leaf senescence as well as abscission, and disease symptom development. Ethylene plays a critical role in the bacterial pathogen Xanthomonas euvesicatoria (X. euvesicatoria)-elicited symptom development in tomato. This protocol describes the measurement of ethylene gas produced by tomato leaves infected with X. euvesicatoria. Infected leaflets are placed in a glass tube for 30 min without sealing. The glass tubes are then capped with a septa stopper, and incubated for an hour...
February 5, 2016: Bio-protocol
https://www.readbyqxmd.com/read/27158639/stable-isotope-resolved-metabolomics-studies-in-ex-vivo-tissue-slices
#20
Teresa W-M Fan, Andrew N Lane, Richard M Higashi
An important component of this methodology is to assess the role of the tumor microenvironment on tumor growth and survival. To tackle this problem, we have adapted the original approach of Warburg (1), by combining thin tissue slices with Stable Isotope Resolved Metabolomics (SIRM) to determine detailed metabolic activity of human tissues. SIRM enables the tracing of metabolic transformations of source molecules such as glucose or glutamine over defined time periods, and is a requirement for detailed pathway tracing and flux analysis...
February 5, 2016: Bio-protocol
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