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Superresolution Microscopy of Drosophila Indirect Flight Muscle Sarcomeres

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Ex vivo Culture Assay Using Human Hair Follicles to Study Circadian Characteristics

Authors: Atsuhiro Nishida, Yoshiki Miyawaki, Koichi Node and Makoto Akashi, date: 06/05/2020, view: 468, Q&A: 0

[Abstract] Ex vivo culture assays of biopsy specimens are advantageous for the experimental evaluation of human circadian characteristics. We developed a simple and non-invasive experimental evaluation method for monitoring the expression of circadian clock genes in an ex vivo culture assay using human hair follicles. This method imposes ...

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: 639, 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 ...

Measurement of Mitotic Spindle Angle and Mitotic Cell Distance in Fixed Tissue of Drosophila Larval Brains

Authors: Maribel Franco and Ana Carmena, date: 11/20/2019, view: 937, Q&A: 0

[Abstract] The positioning and the cleavage plane orientation of mitotic cells in pseudostratified epithelia (PSE) must be tightly regulated since failures in any of these processes might have fatal consequences during development. Here we present a simple method to determine the spindle orientation as well as the positioning of neuroepithelial mitotic cells ...

Ex vivo Drosophila Wing Imaginal Disc Culture and Furin Inhibitor Assay

Authors: Alex Sohr, Lijuan Du and Sougata Roy, date: 08/20/2019, view: 1640, Q&A: 0

[Abstract] Furin is an evolutionarily conserved proprotein convertase (PC) family enzyme with a broad range of substrates that are essential for developmental, homeostatic, and disease pathways. Classical genetic approaches and in vitro biochemical or cell biological assays identified that precursor forms of most growth factor family proteins are ...

Lipid-exchange Rate Assay for Lipid Droplet Fusion in Live Cells

Authors: Jia Wang, Boon Tin Chua, Peng Li and Feng-Jung Chen, date: 07/20/2019, view: 1821, Q&A: 0

[Abstract] Lipid droplets (LDs) are central organelles in maintaining lipid homeostasis. Defective LD growth often results in the development of metabolic disorders. LD fusion and growth mediated by cell death–inducing DNA fragmentation factor alpha (DFFA)-like effector (CIDE) family proteins are crucial for various biological processes including unilocular ...

Determining Oxidative Damage by Lipid Peroxidation Assay in Rat Serum

Authors: Qian Tang, Yu-Wen Su and Cory J. Xian, date: 06/20/2019, view: 2657, Q&A: 0

[Abstract] It has been well-established that malondialdehyde (MDA), which is generated during the process of lipid peroxidation, is a commonly known biomarker for oxidative stress. Therefore, the serum levels of MDA are detected by using the lipid peroxidation assay with commercially available kit to determine the induction of oxidative stress in rat models.

Simultaneous Fluorescent Recordings of Extracellular ATP and Intracellular Calcium in Mammalian Cells

Authors: Nicholas Mikolajewicz and Svetlana V. Komarova, date: 05/20/2019, view: 2229, Q&A: 0

[Abstract] Extracellular ATP is a potent signaling molecule that stimulates intracellular calcium responses through purinergic (P2) receptors in mammalian cells. While extracellular ATP and intracellular calcium can be measured separately, simultaneous monitoring can offer additional insights into P2 receptor physiology. This protocol takes advantage of the ...

Quantification of the Humidity Effect on HR by Ion Leakage Assay

Authors: Musoki Mwimba and Xinnian Dong, date: 04/05/2019, view: 1989, Q&A: 0

[Abstract] We describe a protocol to measure the contribution of humidity on cell death during the effector-triggered immunity (ETI), the plant immune response triggered by the recognition of pathogen effectors by plant resistance genes. This protocol quantifies tissue cell death by measuring ion leakage due to loss of membrane integrity during the ...

High-resolution Immunoelectron Microscopy Techniques for Revealing Distinct Subcellular Type 1 Cannabinoid Receptor Domains in Brain

Authors: Nagore Puente, Itziar Bonilla-Del Río, Svein Achicallende, Patrick C. Nahirney and Pedro Grandes, date: 01/20/2019, view: 3158, Q&A: 0

[Abstract] Activation of type 1 cannabinoid (CB₁) receptors by endogenous, exogenous (cannabis derivatives) or synthetic cannabinoids (i.e., CP 55.940, Win-2) has a wide variety of behavioral effects due to the presence of CB₁ receptors in the brain. In situ hybridization and immunohistochemical techniques have been ...

Cartilage Induction from Mouse Mesenchymal Stem Cells in High-density Micromass Culture

Authors: Takashi Iezaki, Kazuya Fukasawa, Takanori Yamada, Manami Hiraiwa, Katsuyuki Kaneda and Eiichi Hinoi, date: 01/05/2019, view: 2647, Q&A: 0

[Abstract] Mesenchymal stem cells have the ability to differentiate into multiple lineages, including adipocytes, osteoblasts and chondrocytes. Mesenchymal stem cells can be induced to differentiate into chondrocytes in extracellular matrices, such as alginate or collagen gel. Mesenchymal stem cells in a cell pellet or micromass culture can be also induced ...

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