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

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Manganese Superoxide Dismutase Activity Assay in the Yeast Saccharomyces cerevisiae

Authors: Louise Thines and Pierre Morsomme, date: 03/05/2020, view: 785, Q&A: 0

[Abstract] Superoxide dismutases (SODs) act as a primary defence against reactive oxygen species (ROS) by converting superoxide anion radicals (O₂^-) into molecular oxygen (O₂) and hydrogen peroxide (H₂O₂). Members of this enzyme family include CuZnSODs, MnSODs, FeSODs, and NiSODs, depending on the nature ...

Visualizing Filamentous Actin in Chlamydomonas reinhardtii

Authors: Evan W. Craig and Prachee Avasthi, date: 06/20/2019, view: 2178, Q&A: 0

[Abstract] This protocol aims to visualize the filamentous actin network in Chlamydomonas reinhardtii. We improved fixed-cell labeling conditions using the F-actin probe, phalloidin. We created a Chlamydomonas-optimized protocol by halving the phalloidin incubation time, electing for optimal fixation conditions, and selecting for a healthy ...

Measuring Protein Synthesis during Cell Cycle by Azidohomoalanine (AHA) Labeling and Flow Cytometric Analysis

Authors: Koshi Imami and Tomoharu Yasuda, date: 04/20/2019, view: 3074, Q&A: 0

[Abstract] Protein synthesis is one of the most fundamental biological processes to maintain cellular proteostasis. Azidohomoalaine (AHA) is a non-radioactive and “clickable” amino acid analog of methionine which can be incorporated into newly synthesized proteins. Thus, AHA-labeled nascent proteins can be detected and quantified through fluorescent labeling ...

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: 3219, 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 ...

Flow Cytometry Assay for Recycling of LFA-1 in T-lymphocytes

Authors: Katarzyna Potrzebowska, Janne Lehtonen, Malin Samuelsson and Lena Svensson, date: 12/05/2018, view: 2020, Q&A: 0

[Abstract] To enable cells to move forward, cell surface integrins are internalized into an endosomal compartment and subsequently intracellularly transported to be re-exposed at a new site on the cell membrane. Leukocytes are the fastest migrating cell type in the human body, which express the leukocyte-specific integrin LFA-1. Here, we describe a flow ...

Relative Quantitation of Polymerized Actin in Suspension Cells by Flow Cytometry

Authors: Mallory R. Kakley, Katrina B. Velle and Lillian K. Fritz-Laylin, date: 11/20/2018, view: 2652, Q&A: 0

[Abstract] The amount of polymerized actin within a cell can vary widely due to natural processes and/or experimentally induced perturbations. We routinely use this protocol to measure relative polymerized actin content between cell populations by staining cells in suspension with fluorescent phalloidin, then measuring total cell fluorescence using flow ...

Quantifying Podocytes and Parietal Epithelial Cells in Human Urine Using Liquid-based Cytology and WT1 Immunoenzyme Staining

Authors: Hiroyuki Ohsaki, Toru Matsunaga, Taishi Fujita, Yasunori Tokuhara, Shingo Kamoshida and Tadashi Sofue, date: 05/05/2018, view: 3639, Q&A: 0

[Abstract] In glomerular disease, podocytes and parietal epithelial cells (PECs) are shed in the urine. Therefore, urinary podocytes and PECs are noninvasive biomarkers of glomerular disease. The purpose of this protocol is to employ immunocytochemistry to detect podocytes and PECs, using the WT1 antibody on liquid-based cytology slides.

Quantification of Colibactin-associated Genotoxicity in HeLa Cells by In Cell Western (ICW) Using γ-H2AX as a Marker

Authors: Sophie Tronnet and Eric Oswald, date: 03/20/2018, view: 3790, Q&A: 0

[Abstract] The genotoxin colibactin is produced by several species of Enterobacteriaceae. This genotoxin induces DNA damage, cell cycle arrest, senescence and death in eukaryotic cells (Nougayrède et al., 2006; Taieb et al., 2016). Here we describe a method to quantify the genotoxicity of bacteria producing colibactin following a ...

Immunogold Electron Microscopy of the Autophagosome Marker LC3

Authors: Domenico Mattoscio, Andrea Raimondi, Carlo Tacchetti and Susanna Chiocca, date: 12/20/2017, view: 7361, Q&A: 0

[Abstract] Even though autophagy was firstly observed by transmission electron microscopy already in the 1950s (reviewed in Eskelinen et al., 2011), nowadays this technique remains one of the most powerful systems to monitor autophagic processes. The autophagosome, an LC3-positive double membrane structures enclosing cellular materials, represents ...

GFP-Grb2 Translocation Assay Using High-content Imaging to Screen for Modulators of EGFR-signaling

Authors: Julia Petschnigg and Robin Ketteler, date: 09/05/2017, view: 4721, Q&A: 0

[Abstract] High-content screening is a useful tool to understand complex cellular processes and to identify genes, proteins or small molecule compounds that modulate such pathways. High-content assays monitor the function of a protein or pathway by visualizing a change in an image-based readout, such as a change in the localization of a reporter protein. ...

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