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    Differentiation of Myeloid-derived Suppressor Cells from Murine Bone Marrow and Their Co-culture with Splenic Dendritic Cells
    Authors:  Giada Mondanelli and Claudia Volpi, date: 09/20/2017, view: 21, Q&A: 0
    Myeloid-derived suppressor cells (MDSCs) possess the ability to suppress the immune response, and to amplify the regulatory properties of other immune cells, i.e., dendritic cells. Here we describe a protocol in which MDSCs were ...
    Phagocytosis Assay of Necroptotic Cells by Cardiac Myofibroblasts
    In myocardial infarction (MI), a plenty of cardiomyocytes undergo necrosis and necroptosis due to the lack of oxygen and nutrients. The dead cardiomyocytes are promptly engulfed by phagocytes. When the dead cells are not engulfed, the noxious ...
    An Assay to Determine Phagocytosis of Apoptotic Cells by Cardiac Macrophages and Cardiac Myofibroblasts
    In myocardial infarction (MI), a number of cardiomyocytes undergo apoptosis. These apoptotic cardiomyocytes are promptly engulfed by phagocytes. If the dead cells are not engulfed, their noxious contents are released outside, resulting in induction ...
    Stereotaxic Adeno-associated Virus Injection and Cannula Implantation in the Dorsal Raphe Nucleus of Mice
    Authors:  Patrícia A. Correia, Sara Matias and Zachary F. Mainen, date: 09/20/2017, view: 18, Q&A: 0
    Optogenetic methods are now widespread in neuroscience research. Here we present a detailed surgical procedure to inject adeno-associated viruses and implant optic fiber cannulas in the dorsal raphe nucleus (DRN) of living mice. Combined with ...
    Preparation of Primary Cultures of Embryonic Rat Hippocampal and Cerebrocortical Neurons
    This protocol aims at standardizing the procedure to obtain primary cultures of hippocampal and cerebrocortical neurons for in vitro experiments. Cultures should be prepared from cells isolated during embryonic development when neuronal ...
    Uptake Assays to Monitor Anthracyclines Entry into Mammalian Cells
    Authors:  Nicolas Brosseau, Emil Andreev and Dindial Ramotar, date: 09/20/2017, view: 14, Q&A: 0
    Anthracyclines, such as doxorubicin and daunorubicin, are DNA damaging agents that autofluoresce and can be readily detected in cells. Herein, we developed suitable assays to quantify and localize daunorubicin in mammalian cells. These assays can be ...
    Differentiation of Myeloid-derived Suppressor Cells from Murine Bone Marrow and Their Co-culture with Splenic Dendritic Cells
    Authors:  Giada Mondanelli and Claudia Volpi, date: 09/20/2017, view: 21, Q&A: 0
    [Abstract] Myeloid-derived suppressor cells (MDSCs) possess the ability to suppress the immune response, and to amplify the regulatory properties of other immune cells, i.e., dendritic cells. Here we describe a protocol in which MDSCs were differentiated from murine bone marrow cells, and CD11c+ dendritic cells were purified from murine ...
    Phagocytosis Assay of Necroptotic Cells by Cardiac Myofibroblasts
    [Abstract] In myocardial infarction (MI), a plenty of cardiomyocytes undergo necrosis and necroptosis due to the lack of oxygen and nutrients. The dead cardiomyocytes are promptly engulfed by phagocytes. When the dead cells are not engulfed, the noxious contents of the cells are released outside, and thus, induce inflammation, and obstruct the function of ...
    An Assay to Determine Phagocytosis of Apoptotic Cells by Cardiac Macrophages and Cardiac Myofibroblasts
    [Abstract] In myocardial infarction (MI), a number of cardiomyocytes undergo apoptosis. These apoptotic cardiomyocytes are promptly engulfed by phagocytes. If the dead cells are not engulfed, their noxious contents are released outside, resulting in induction of inflammation. Therefore, the removal of these dead cells is necessary. However, the contribution ...
    Stereotaxic Adeno-associated Virus Injection and Cannula Implantation in the Dorsal Raphe Nucleus of Mice
    Authors:  Patrícia A. Correia, Sara Matias and Zachary F. Mainen, date: 09/20/2017, view: 18, Q&A: 0
    [Abstract] Optogenetic methods are now widespread in neuroscience research. Here we present a detailed surgical procedure to inject adeno-associated viruses and implant optic fiber cannulas in the dorsal raphe nucleus (DRN) of living mice. Combined with transgenic mouse lines, this protocol allows specific targeting of serotonin-producing neurons in the ...
    Preparation of Primary Cultures of Embryonic Rat Hippocampal and Cerebrocortical Neurons
    [Abstract] This protocol aims at standardizing the procedure to obtain primary cultures of hippocampal and cerebrocortical neurons for in vitro experiments. Cultures should be prepared from cells isolated during embryonic development when neuronal precursor cells are not yet fully differentiated. This helps increasing the quality and quantity of ...
    Uptake Assays to Monitor Anthracyclines Entry into Mammalian Cells
    Authors:  Nicolas Brosseau, Emil Andreev and Dindial Ramotar, date: 09/20/2017, view: 14, Q&A: 0
    [Abstract] Anthracyclines, such as doxorubicin and daunorubicin, are DNA damaging agents that autofluoresce and can be readily detected in cells. Herein, we developed suitable assays to quantify and localize daunorubicin in mammalian cells. These assays can be exploited to identify components that are involved in the uptake of anthracyclines.
    Arginine-rich Peptides Can Actively Mediate Liquid-liquid Phase Separation
    Authors:  Steven Boeynaems, Mathias De Decker, Peter Tompa and Ludo Van Den Bosch, date: 09/05/2017, view: 372, Q&A: 0
    [Abstract] Studying liquid-liquid phase separation (LLPS) of proteins provides key insights into the biogenesis of membraneless organelles and pathological protein aggregation in disease. We have established a protocol for inducing the phase separation of arginine-rich peptides, which allows for studying their molecular determinants and dynamics (Boeynaems ...
    Peroxisome Motility Measurement and Quantification Assay
    Authors:  Jeremy Metz, Inês G. Castro and Michael Schrader, date: 09/05/2017, view: 178, Q&A: 0
    [Abstract] Organelle movement, distribution and interaction contribute to the organisation of the eukaryotic cell. Peroxisomes are multifunctional organelles which contribute to cellular lipid metabolism and ROS homeostasis. They distribute uniformly in mammalian cells and move along microtubules via kinesin and dynein motors. Their metabolic cooperation ...
    Expression and Purification of a Mammalian P2X7 Receptor from Sf9 Insect Cells
    Authors:  Akira Karasawa and Toshimitsu Kawate, date: 09/05/2017, view: 314, Q&A: 0
    [Abstract] The P2X7 receptor is an extracellular ATP-gated ion channel found only in eukaryotes (Bartlett et al., 2014). Due to its unique properties among P2X receptors, such as formation of a large conductance pore, the P2X7 receptor has been implicated in devastating diseases like chronic pain (North and Jarvis, 2013). However, mechanisms ...
    Protocol for Establishing a Multiplex Image-based Autophagy RNAi Screen in Cell Cultures
    Authors:  Jennifer Jung and Christian Behrends, date: 09/05/2017, view: 224, Q&A: 0
    [Abstract] Autophagy is a recycling pathway, in which intracellular cargoes including protein aggregates and bacteria are engulfed by autophagosomes and subsequently degraded after fusion with lysosomes. Dysregulation of this process is involved in several human diseases such as cancer or neurodegeneration. Hence, advancing our understanding of how autophagy ...