Mammalia

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    Protocols in Current Issue
    Rapid Nickel-based Isolation of Extracellular Vesicles from Different Biological Fluids
    [Abstract] Extracellular vesicles (EVs) are membranous structures that cells massively release in extracellular fluids. EVs are cargo of cellular components such as lipids, proteins, and nucleic acids that can work as a formidable source in liquid biopsy studies searching for disease biomarkers. We recently demonstrated that nickel-based isolation (NBI) is a ...
    Identification of Target Protein for Bio-active Small Molecule Using Photo-cross Linked Beads and MALDI-TOF Mass Spectrometry
    [Abstract] Development of methods for protein identification is one of the important aspects of proteomics. Here, we report a protocol for the preparation of compound conjugated beads by photo-crosslinking, affinity purification, gel electrophoresis, and highly sensitive mass spectrometric assay for drug-target identification. Although there are several ...
    Automated Analysis of Cell Surface Ruffling: Ruffle Quantification Macro
    Authors:  Nicholas D. Condon, Jennifer L. Stow and Adam A. Wall, date: 01/20/2020, view: 285, Q&A: 0
    [Abstract] Cell surface protrusions include F-actin rich, wave-like ruffles that are erected transiently in response to stimuli and during cell migration. Macrophages are innate immune cells that ruffle constitutively and more dramatically in cells activated by pathogens. Dorsal ruffles and their resulting macropinosomes are key sites for environmental ...
    Quantification of Nitric Oxide and Reactive Oxygen Species in Leishmania-infected J774A.1 Macrophages as a Response to the in vitro Treatment with a Natural Product Compound
    Authors:  Kalliopi Karampetsou, Olga S. Koutsoni and Eleni Dotsika, date: 12/05/2019, view: 472, Q&A: 0
    [Abstract] Leishmaniasis is a parasitic disease caused by the obligatory intracellular protozoa Leishmania spp. Current therapeutic options are limited and thus, drug discovery against leishmaniasis is very important. Nevertheless, there is a great difficulty to develop therapeutic strategies against the disease because the parasite deploys various ...
    Mapping the Mechanome–A Protocol for Simultaneous Live Imaging and Quantitative Analysis of Cell Mechanoadaptation and Ingression
    [Abstract] Mechanomics, the mechanics equivalent of genomics, is a burgeoning field studying mechanical modulation of stem cell behavior and lineage commitment. Analogous to mechanical testing of a living material as it adapts and evolves, mapping of the mechanome necessitates the development of new protocols to assess changes in structure and function in ...
    ELISA Based Protein Ubiquitylation Measurement
    Authors:  Yuka Kamada, Ryosuke Fukuda and Tsukasa Okiyoneda, date: 11/20/2019, view: 656, Q&A: 0
    [Abstract] Ubiquitylation is a common post-translational modification of cellular proteins that results in proteasomal and lysosomal degradations. Ubiquitylation is generally measured by methods such as immunoblotting using anti-ubiquitin antibodies after isolating the protein-of-interest by denaturing immunoprecipitation. The following protocol can be used ...
    Differentiation of Mouse Embryonic Stem Cells to Neuronal Cells Using Hanging Droplets and Retinoic Acid
    Authors:  Jeroen Witteveldt and Sara Macias, date: 11/05/2019, view: 586, Q&A: 0
    [Abstract] Controlled differentiation of embryonic stem cells is an essential tool in stem cell research. In this protocol, we describe a simple differentiation protocol involving the induction of embryoid body formation in mouse embryonic stem cells (mESC) using hanging droplets, followed by differentiation into a neuronal lineage.
    In vitro Screening of Antileishmanial Activity of Natural Product Compounds: Determination of IC50, CC50 and SI Values
    Authors:  Olga S. Koutsoni, Kalliopi Karampetsou and Eleni Dotsika, date: 11/05/2019, view: 428, Q&A: 0
    [Abstract] Neglected tropical diseases gain the scientific interest of numerous research programs in an attempt to achieve their effective control or elimination. In this attempt, more cutting-edge public health policies and research are needed for the discovery of new, safer and effective drugs originated from natural products. Here, we describe protocols ...
    Isolation and Transcriptomic Profiling of Single Myofibers from Mice
    [Abstract] Skeletal muscle is composed of different cells and myofiber types, with distinct metabolic and structural features. Generally, transcriptomic analysis of skeletal muscle is performed using whole muscle, resulting in average information as all cells composing the organ contribute to the expression value detected for each gene with the loss of ...
    Quantitation of TLR4 Internalization in Response to LPS in Thioglycollate Elicited Peritoneal Mouse Macrophages by Flow Cytometry
    Authors:  Katharina Richard, Stefanie N. Vogel and Darren J. Perkins, date: 09/20/2019, view: 601, Q&A: 0
    [Abstract] Internalization of cell surface Toll-like Receptor 4 (TLR4) is a convenient read-out to measure LPS dependent activation of the TRIF adaptor pathway. We here provide a protocol to quantify the LPS dependent internalization of TLR4 using thioglycollate-elicited peritoneal macrophages by flow cytometry.



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