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Non-separate Mouse Sclerochoroid/RPE/Retina Staining and Whole Mount for the Integral Observation of Subretinal Layer

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Quantification of the Surface Expression of G Protein-coupled Receptors Using Intact Live-cell Radioligand Binding Assays

Authors: Xin Xu and Guangyu Wu, date: 09/20/2020, view: 1341, Q&A: 0

[Abstract] G protein-coupled receptors (GPCRs) are the most structurally diverse family of signaling proteins and regulate a variety of cell function. For most GPCRs, the cell surface is their functional destination where they are able to respond a wide range of extracellular stimuli, leading to the activation of intracellular signal transduction cascades. ...

Production and Bioassay of a Diffusible Factor that Induces Gametophyte-to-Sporophyte Developmental Reprogramming in the Brown Alga Ectocarpus

Authors: Haiqin Yao, Delphine Scornet, Yacine Badis, Akira F. Peters, Murielle Jam, Cecile Hervé, Philippe Potin, Susana M. Coelho and J. Mark Cock, date: 09/20/2020, view: 1381, Q&A: 0

[Abstract] The brown alga Ectocarpus has a haploid-diploid life cycle that involves alternation between two multicellular generations, the sporophyte and the gametophyte. Life cycle generation is not determined by ploidy but by a genetic system that includes two different three amino acid loop extension homeodomain transcription factors called ...

Flow-cytometric Detection of Low-level Reactive Oxygen Species in Cell Lines and Primary Immune Cells

Authors: Kevin Bode, Corinna Link, Peter H. Krammer and Heiko Weyd, date: 09/05/2020, view: 2112, Q&A: 0

[Abstract] Depending on its concentration and cellular origin the production of reactive oxygen species (ROS) in the organism serves a variety of functions. While high concentrations during an oxidative burst are used to fight pathogens, low to moderate amounts of ROS act as signaling molecules important for several physiological processes such as regulation ...

Assessing Gα_q/15-signaling with IP-One: Single Plate Transfection and Assay Protocol for Cell-Based High-Throughput Assay

Authors: Élie Besserer-Offroy, Rebecca L Brouillette, Jean-Michel Longpré and Philippe Sarret, date: 08/20/2020, view: 1023, Q&A: 0

[Abstract] Cell-based functional assays are an important part of compound screening and drug lead optimization, and they can also play a crucial role in the determination of the residues involved in ligand binding and signaling for a particular G-protein-coupled receptor. Conventional methods used for Gα_q/15-coupled receptors rely on the use of ...

Organotypic Slice Culture of the Embryonic Mouse Brain

Authors: James M. Clegg and Thomas Pratt, date: 07/05/2020, view: 962, Q&A: 0

[Abstract] Organotypic slice culture is a powerful technique for exploring the embryonic development of the mammalian brain. In this protocol we describe a basic slice culture technique we have used for two sets of experiments: axon guidance transplant assays and bead culture assays.

Quantification of Protein Kinase A (PKA) Activity by An in vitro Radioactive Assay Using the Mouse Sperm Derived Enzyme

Authors: Cintia Stival, Carolina Baro Graf, Pablo E. Visconti and Dario Krapf, date: 06/20/2020, view: 1365, Q&A: 0

[Abstract] In order to acquire fertilizing potential, mammalian sperm must undergo a process known as capacitation, which relies on the early activation of Protein Kinase A (PKA). Frequently, PKA activity is assessed in whole-cell experiments by analyzing the phosphorylation status of its substrates in a western-blot. This technique ...

FRET Reporter Assays for cAMP and Calcium in a 96-well Format Using Genetically Encoded Biosensors Expressed in Living Cells

Authors: Brandon T. Milliken, Robert P. Doyle, George G. Holz and Oleg G. Chepurny, date: 06/05/2020, view: 1861, Q&A: 0

[Abstract] Stimulation of G protein-coupled receptors (GPCR) by hormones and neurotransmitters elicits cellular responses, many of which result from alterations in the concentrations of cytosolic cAMP and Ca²⁺. Here, we describe a microplate reader fluorescence resonance energy transfer (FRET) assay that uses the genetically encoded biosensors ...

An ex vivo Approach to Assess Mitochondrial ROS by Flow Cytometry in AAV-tagged Astrocytes in Adult Mice

Authors: Carlos Vicente-Gutierrez and Juan P. Bolaños, date: 03/20/2020, view: 1718, Q&A: 0

[Abstract] Mitochondrial reactive oxygen species (mROS) are naturally produced signalling molecules extremely relevant for understanding both health- and disease-associated biological processes. The study of mROS in the brain is currently underway to decipher their physiopathological roles and contributions in neurological diseases. Recent advances in this ...

Insect Feeding Assays with Spodoptera exigua on Arabidopsis thaliana

Authors: Yanrong You, Chunpeng An and Chuanyou Li, date: 03/05/2020, view: 1573, Q&A: 0

[Abstract] Plant-insect interaction is an important field for studying plant immunity. The beet armyworm, Spodoptera exigua, is one of the best-known agricultural pest insects and is usually used to study plant interactions with chewing insects. Here, we describe a protocol for insect feeding assays with Spodoptera exigua lavae using model ...

Optogenetic Tuning of Ligand Binding to The Human T cell Receptor Using The opto-ligand-TCR System

Authors: O. Sascha Yousefi, Maximilian Hörner, Maximilian Wess, Vincent Idstein, Wilfried Weber and Wolfgang W. A. Schamel, date: 03/05/2020, view: 1797, Q&A: 0

[Abstract] T cells are one major cell type of the immune system that use their T cell antigen receptor (TCR) to bind and respond to foreign molecules derived from pathogens. The ligand-TCR interaction half-lives determine stimulation outcome. Until recently, scientists relied on mutating either the TCR or its ligands to investigate how varying TCR-ligand ...

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