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Hypochlorous Acid Staining with R19-S in the Drosophila Intestine upon Ingestion of Opportunistic Bacteria

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Protocol to Quantitatively Assess the Structural Integrity of Perineuronal Nets ex vivo

Authors: Bhanu P. Tewari and Harald Sontheimer, date: 05/20/2019, view: 212, Q&A: 0

[Abstract] Perineuronal nets (PNNs) are extracellular matrix assemblies of highly negatively charged proteoglycans that wrap around fast-spiking parvalbumin (PV) expressing interneurons in the cerebral cortex. PNNs play important roles in neuronal plasticity and modulate biophysical properties of the enclosed interneurons. Various central nervous system ...

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: 554, 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: 910, 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: 921, 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: 1215, 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 ...

Staining the Germline in Live Caenorhabditis elegans: Overcoming Challenges by Applying a Fluorescent-dye Feeding Strategy

Author: K. Adam Bohnert, date: 11/05/2018, view: 1200, Q&A: 0

[Abstract] C. elegans provides a tractable model organism for studying germline cell biology. Microscopy experiments are relatively facile, as this worm is transparent and germline development can be observed in real-time using DIC microscopy and/or fluorescent transgenes. Despite these many tools, robust staining techniques for imaging germ cells ...

Assessing Membrane Fluidity and Visualizing Fluid Membrane Domains in Bacteria Using Fluorescent Membrane Dyes

Authors: Michaela Wenzel, Norbert O. E. Vischer, Henrik Strahl and Leendert W. Hamoen, date: 10/20/2018, view: 1314, Q&A: 0

[Abstract] Membrane fluidity is a key parameter of bacterial membranes that undergoes quick adaptation in response to environmental challenges and has recently emerged as an important factor in the antibacterial mechanism of membrane-targeting antibiotics. The specific level of membrane fluidity is not uniform across the bacterial cell membrane. Rather, ...

Artificial Inoculation of Epichloë festucae into Lolium perenne, and Visualisation of Endophytic and Epiphyllous Fungal Growth

Authors: Yvonne Becker, Kimberly A. Green, Barry Scott and Matthias Becker, date: 09/05/2018, view: 1447, Q&A: 0

[Abstract] Natural hosts for the fungal endophyte Epichloë festucae include Festuca rubra (fine fescue) and Festuca trachyphylla (hard fescue). Some strains also form stable associations with Lolium perenne (perennial ryegrass). L. perenne is a suitable host to study fungal endophyte–grass interactions, such as endophytic ...

Visualization of RNA at the Single Cell Level by Fluorescent in situ Hybridization Coupled to Flow Cytometry

Authors: Alice Coillard and Elodie Segura, date: 06/20/2018, view: 2115, Q&A: 0

[Abstract] The protocol described here has been developed to detect RNA at the single cell level. Fluorescent probes hybridize to target RNAs and are detected by flow cytometry after multiple amplification steps. Different types of RNA can be detected such as mRNA, long noncoding RNA, viral RNA or telomere RNA and up to 4 different target probes can be used ...

Osteoblast Sorting and Intracellular Staining of CXCL12

Authors: Weihuan Wang, Gurnoor Majhail, Cui Lui and Lan Zhou, date: 05/20/2018, view: 2176, Q&A: 0

[Abstract] Osteoblasts are bone marrow endosteum-lining niche cells playing important roles in the regulation of hematopoietic stem cells by secreting factors and cell adhesion molecules. Characterization of primary osteoblasts has been achieved through culture of outgrowth of collagenase treated bone. Immunophenotyping and flow-based analysis of long bone ...

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