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Preparation of Drosophila Polytene Chromosomes, Followed by Immunofluorescence Analysis of Chromatin Structure by Multi-fluorescence Correlations

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Organotypic Slice Culture of the Embryonic Mouse Brain

Authors: James M. Clegg and Thomas Pratt, date: 07/05/2020, view: 256, 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: 596, 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: 687, 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: 869, 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: 795, 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: 987, 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 ...

Phospho-protein Analysis in Adherent Cells Using Flow Cytometry

Authors: Renu Sharma, Amit Sharma, Atul Kumar and Bithiah Grace Jaganathan, date: 10/20/2019, view: 1410, Q&A: 0

[Abstract] Protein phosphorylation is one of the most important post-translational modifications, which acts as a reversible on or off switch for the activity of a large number of proteins. Analyzing the phosphorylation status of different proteins can reveal the alterations in the state of the cells in response to cellular damage, cancer and pharmaceutical ...

Tandem Affinity Purification of SBP-CBP-tagged Type Three Secretion System Effectors

Authors: Laura Berneking, Marie Schnapp, Theresa Nauth and Moritz Hentschke, date: 06/20/2019, view: 1622, Q&A: 0

[Abstract] Identification of protein-protein interactions of bacterial effectors and cellular targets during infection is at the core to understand how bacteria manipulate the infected host to overcome the immune response. Potential interacting proteins might be identified by genetic methods, i.e., two hybrid screens and could be verified by ...

Using Arabidopsis Mesophyll Protoplasts to Study Unfolded Protein Response Signaling

Authors: Yan Bao and Diane C. Bassham, date: 12/05/2018, view: 2804, Q&A: 0

[Abstract] Various environmental stresses or artificial reagents can trigger unfolded protein accumulation in the endoplasmic reticulum (ER) due to the folding capacity of the ER being exceeded. This is termed ER stress, and triggers the unfolded protein response (UPR). Assays for activation of the UPR in plants include Tunicamycin (Tm)- or dithiothreitol ...

Measurement of Dopamine Using Fast Scan Cyclic Voltammetry in Rodent Brain Slices

Authors: Madelyn I. Mauterer, Paige M. Estave, Katherine M. Holleran and Sara R. Jones, date: 10/05/2018, view: 4871, Q&A: 0

[Abstract] Fast scan cyclic voltammetry (FSCV) is an electrochemical technique that allows sub-second detection of oxidizable chemical species, including monoamine neurotransmitters such as dopamine, norepinephrine, and serotonin. This technique has been used to record the physiological dynamics of these neurotransmitters in brain tissue, including their ...

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