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Shruti Thapliyal, Kavita Babu
Pentylenetetrazole (PTZ) is a GABAA receptor antagonist and is used to monitor presynaptic defects in the release of the inhibitory neurotransmitter GABA. PTZ is a competitive inhibitor of GABA, and prevents binding of GABA on the GABAA receptors present on the surface of muscle. In the absence of GABA binding, the excitatory to inhibitory signal ratio increases resulting in a convulsive phenotype. This assay provides a fast and reliable method to detect presynaptic defects in GABAergic synaptic transmission...
September 5, 2018: Bio-protocol
Elizabeth A Fucich, David A Morilak
Maladaptive avoidance behaviors are seen in many stress-related psychiatric illnesses. Patients with these illnesses favor passive, avoidant coping strategies rather than adaptive, active coping strategies. Preclinically, coping strategy can be measured in rats using the shock-probe defensive burying test, wherein rats receive a shock from an electrified probe inserted into a test cage that mimics their home cage environment, and behavioral output (immobility or burying) is recorded for 15 min following the shock...
September 5, 2018: Bio-protocol
Guo Chen, Xingming Deng
Cell synchronization is widely used in studying mechanisms involves in regulation of cell cycle progression. Through synchronization, cells at distinct cell cycle stage could be obtained. Thymidine is a DNA synthesis inhibitor that can arrest cell at G1/S boundary, prior to DNA replication. Here, we present the protocol to synchronize cells at G1/S boundary by using double thymidine block. After release into normal medium, cell population at distinct cell cycle phase could be collected at different time points...
September 5, 2018: Bio-protocol
Elena Redondo-Castro, Catriona J Cunningham, Jonjo Miller, Stuart A Cain, Stuart M Allan, Emmanuel Pinteaux
The 3D culture of human mesenchymal stem cells (hMSCs) represents a more physiological environment than classical 2D culture and has been used to enhance the MSC secretome or extend cell survival after transplantation. Here we describe a simple and affordable method to generate 3D spheroids of hMSCs by seeding them at high density in a low-binding 96-well plate. Spheroids of hMSCs cultured in low-binding 96-well plates can be used to study the basic biology of the cells and to generate conditioned media or spheroids to be used in transplantation therapeutic approaches...
August 20, 2018: Bio-protocol
Jeffrey W Perry, Andrew W Tai
Protein tagging is a powerful method of investigating protein function. However, modifying positive-strand RNA virus proteins in the context of viral infection can be particularly difficult as their compact genomes and multifunctional proteins mean even small changes can inactivate or attenuate the virus. Although targeted approaches to functionally tag viral proteins have been successful, these approaches are time consuming and inefficient. A strategy that has been successfully applied to several RNA viruses is whole-genome transposon insertional mutagenesis...
August 20, 2018: Bio-protocol
Natalia Cattelan, Osvaldo Miguel Yantorno, Rajendar Deora
No abstract text is available yet for this article.
August 5, 2018: Bio-protocol
Miranda Sc Wilson, Adolfo Saiardi
Inositol phosphates (IPs) comprise a family of ubiquitous eukaryotic signaling molecules. They have been linked to the regulation of a pleiotropy of important cellular activities, but low abundance and detection difficulties have hampered our understanding. Here we present a method to purify and enrich IPs or other phosphate-rich metabolites from mammalian cells or other sample types. Acid-extracted IPs from cells bind selectively via their phosphate groups to titanium dioxide beads. After washing, the IPs are easily eluted from the beads by increasing the pH...
August 5, 2018: Bio-protocol
Rachel Fellows, Patrick Varga-Weisz
Class I histone deacetylases (HDACs) are efficient histone decrotonylases, broadening the enzymatic spectrum of these important (epi-)genome regulators and drug targets. Here, we describe an in vitro approach to assaying class I HDACs with different acyl-histone substrates, including crotonylated histones and expand this to examine the effect of inhibitors and estimate kinetic constants.
July 20, 2018: Bio-protocol
Alexa J Roemmich, Soleil S Schutte, Diane K O'Dowd
Cost-effective and efficient, the fruit fly ( Drosophila melanogaster ) has been used to make many key discoveries in the field of neuroscience and to model a number of neurological disorders. Great strides in understanding have been made using sophisticated molecular genetic tools and behavioral assays. Functional analysis of neural activity was initially limited to the neuromuscular junction (NMJ) and in the central nervous system (CNS) of embryos and larvae. Elucidating the cellular mechanisms underlying neurological processes and disorders in the mature nervous system have been more challenging due to difficulty in recording from neurons in adult brains...
July 20, 2018: Bio-protocol
Guilherme C de Oliveira, Karina S Kanamori, Maria Auxiliadora-Martins, Claudia C S Chini, Eduardo N Chini
CD38 is a multifunctional enzyme involved in calcium signaling and Nicotinamide Adenine Dinucleotide (NAD+ ) metabolism. Through its major activity, the hydrolysis of NAD+ , CD38 helps maintain the appropriate levels of this molecule for all NAD+ -dependent metabolic processes to occur. Due to current advances and studies relating NAD+ decline and the development of multiple age-related conditions and diseases, CD38 gained importance in both basic science and clinical settings. The discovery and development of strategies to modulate its function and, possibly, treat diseases and improve health span put CD38 under the spotlights...
July 20, 2018: Bio-protocol
Michael S Schappe, Bimal N Desai
After recognizing extracellular bacterial lipopolysaccharide (LPS), the toll-like receptor 4 (TLR4)-CD14 signaling complex initiates two distinct signaling pathways-one from the plasma membrane and the other from the signaling endosomes (Kagan et al., 2008). Understanding the early stages of TLR4 signal transduction therefore requires a robust and quantitative method to measure LPS-triggered TLR4 and CD14 receptor endocytosis, one of the earliest events of LPS detection. Here, we describe a flow cytometry-based method that we used recently to study the role of the ion channel TRPM7 in TLR4 endocytosis (Schappe et al...
July 20, 2018: Bio-protocol
Karina S Kanamori, Guilherme C de Oliveira, Maria Auxiliadora-Martins, Renee A Schoon, Joel M Reid, Eduardo N Chini
Current studies on the age-related development of metabolic dysfunction and frailty are each day in more evidence. It is known, as aging progresses, nicotinamide adenine dinucleotide (NAD+ ) levels decrease in an expected physiological process. Recent studies have shown that a reduction in NAD+ is a key factor for the development of age-associated metabolic decline. Increased NAD+ levels in vivo results in activation of pro-longevity and health span-related factors. Also, it improves several physiological and metabolic parameters of aging, including muscle function, exercise capacity, glucose tolerance, and cardiac function in mouse models of natural and accelerated aging...
July 20, 2018: Bio-protocol
Alina C Teuscher, Collin Y Ewald
Green fluorescent protein (GFP) is widely used as a molecular tool to assess protein expression and localization. In C. elegans , the signal from weakly expressed GFP fusion proteins is masked by autofluorescence emitted from the intestinal lysosome-related gut granules. For instance, the GFP fluorescence from SKN-1 transcription factor fused to GFP is barely visible with common GFP (FITC) filter setups. Furthermore, this intestinal autofluorescence increases upon heat stress, oxidative stress (sodium azide), and during aging, thereby masking GFP expression even from proximal tissues...
July 20, 2018: Bio-protocol
Michael M Wördehoff, Wolfgang Hoyer
Studying the aggregation of amyloid proteins like α-synuclein in vitro is a convenient and popular tool to gain kinetic insights into aggregation as well as to study factors ( e.g. , aggregation inhibitors) that influence it. These aggregation assays typically make use of the fluorescence dye Thioflavin T as a sensitive fluorescence reporter of amyloid fibril formation and are conducted in a plate-reader-based format, permitting the simultaneous screening of multiple samples and conditions. However, aggregation assays are generally prone to poor reproducibility due to the stochastic nature of fibril nucleation and the multiplicity of modulating factors...
July 20, 2018: Bio-protocol
Andrew D Doyle
Rat tail collagen solutions have been used as polymerizable in vitro three-dimensional (3D) extracellular matrix (ECM) gels for single and collective cell migration assays as well as spheroid formation. These 3D hydrogels are a relatively inexpensive, simple to use model system that can mimic the in vivo physical characteristics of numerous tissues within the body, namely the skin. While confocal imaging techniques such as fluorescence reflection and two-photon microscopy are able to visualize collagen fibrils during 3D imaging without fluorescence, other imaging modalities require direct conjugation of fluorescent dyes to collagen...
July 5, 2018: Bio-protocol
Yolanda Schaerli
High-throughput screening of a DNA library expressed in a bacterial population for identifying potentially rare members displaying a property of interest is a crucial step for success in many experiments such as directed evolution of proteins and synthetic circuits and deep mutational scanning to identify gain- or loss-of-function mutants. Here, I describe a protocol for high-throughput screening of bacterial ( E. coli ) microcolonies in gel beads. Single cells are encapsulated into monodisperse water-in-oil emulsion droplets produced with a microfluidic device...
July 5, 2018: Bio-protocol
Sabrina Flütsch, Luca Distefano, Diana Santelia
In this protocol, we describe how to quantify starch in guard cells of Arabidopsis thaliana using the fluorophore propidium iodide and confocal laser scanning microscopy. This simple method enables monitoring, with unprecedented resolution, the dynamics of starch in guard cells.
July 5, 2018: Bio-protocol
Kate Kosmac, Bailey D Peck, R Grace Walton, Jyothi Mula, Philip A Kern, Marcas M Bamman, Richard A Dennis, Cale A Jacobs, Christian Lattermann, Darren L Johnson, Charlotte A Peterson
Macrophages have well-characterized roles in skeletal muscle repair and regeneration. Relatively little is known regarding the role of resident macrophages in skeletal muscle homeostasis, extracellular matrix remodeling, growth, metabolism and adaptation to various stimuli including exercise and training. Despite speculation into macrophage contributions during these processes, studies characterizing macrophages in non-injured muscle are limited and methods used to identify macrophages vary. A standardized method for the identification of human resident skeletal muscle macrophages will aide in the characterization of these immune cells and allow for the comparison of results across studies...
June 20, 2018: Bio-protocol
Ben Niu, Roberto Kolter
Besides analyzing the composition and dynamics of microbial communities, plant microbiome research aims to understanding the mechanism of plant microbiota assembly and their biological functions. Here, we describe procedures to investigate the role of bacterial interspecies interactions in root microbiome assembly and the beneficial effects of the root microbiota on hosts by using a maize root-associated simplified seven-species ( Stenotrophomonas maltophilia , Ochrobactrum pituitosum , Curtobacterium pusillum , Enterobacter cloacae , Chryseobacterium indologenes , Herbaspirillum frisingense and Pseudomonas putida ) synthetic bacterial community described in our previous work...
June 20, 2018: Bio-protocol
Jie Zhu, Deepak A Lamba
Retinal degeneration leads to loss of light-sensing photoreceptors eventually resulting in vision impairment and impose a heavy burden on both patients and the society. Currently available treatment options are very limited and mainly palliative. Ever since the discovery of human pluripotent stem cell technologies, cell replacement therapy has become a promising therapeutic strategy for these patients and may help restore visual function. Reproducibly generating enriched retinal cells including retinal progenitors and differentiated retinal neurons such as photoreceptors using human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells in a dish is an essential first step for developing stem cell-based therapies...
June 20, 2018: Bio-protocol
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