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Mammalia

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    Isolation of Primary Human Skeletal Muscle Cells
    Authors:  Janelle M. Spinazzola and Emanuela Gussoni, date: 11/05/2017, view: 177, Q&A: 0
    Primary myoblast culture is a valuable tool in research of muscle disease, pathophysiology, and pharmacology. This protocol describes techniques for dissociation of cells from human skeletal muscle biopsies and enrichment for a highly myogenic ...
    In vitro NLK Kinase Assay
    Authors:  Sungho Moon, Jiyoung Kim and Eek‐hoon Jho, date: 11/05/2017, view: 187, Q&A: 0
    This protocol provides step by step instructions to perform an in vitro kinase assay for nemo-like kinase. In addition, this protocol also describes an efficient method using mild lysis buffer for expression and purification of Glutathione ...
    Assessment of Aversion of Acute Pain Stimulus through Conditioned Place Aversion
    Pain is a complex experience. The aversive component of pain has been assessed through conditioned place aversion in rodents. However, this behavioral test does not allow the evaluation of the aversion of an acute pain stimulus. In Zhang et al. ...
    Histochemical Staining of Collagen and Identification of Its Subtypes by Picrosirius Red Dye in Mouse Reproductive Tissues
    Collagen is one of the foremost components of tissue extracellular matrix (ECM). It provides strength, elasticity and architecture to the tissue enabling it to bear the wear and tear from external factors like physical stress as well as internal ...
    Isolation, Culture and Differentiation of Adult Hippocampal Precursor Cells
    Authors:  Stefanie N. Bernas, Odette Leiter, Tara L. Walker and Gerd Kempermann, date: 11/05/2017, view: 370, Q&A: 0
    There are two neurogenic niches in the adult mammalian brain: the subventricular zone of the lateral ventricle and the subgranular zone of the hippocampal dentate gyrus. Cells from these areas can be isolated and maintained in vitro, using ...
    Monitoring the Targeting of Cathepsin D to the Lysosome by Metabolic Labeling and Pulse-chase Analysis
    Authors:  Lucas A. Tavares and Luis L. P. daSilva, date: 11/05/2017, view: 220, Q&A: 0
    Mannose 6-phosphate receptors function can be studied in living cells by investigating alterations in processing and secretion of their ligand Cathepsin D. The assay described here is well established in the literature and comprises the metabolic ...
    Proximal Ligation Assay (PLA) on Lung Tissue and Cultured Macrophages to Demonstrate Protein-protein Interaction
    Authors:  Roberto Mendez and Santanu Banerjee, date: 11/05/2017, view: 182, Q&A: 0
    In this protocol, we describe proximal ligation assay (PLA), an antibody-based detection method for protein-protein interaction. This method relies on specific binding of individual primary antibodies to the two putative interacting proteins. The ...
    Xenograft Mouse Model of Human Uveal Melanoma
    Authors:  Yao Chen, Xiao Liu, Ling Gao and Yongqing Liu, date: 11/05/2017, view: 161, Q&A: 0
    Uveal melanoma (UM) is a malignant intraocular tumor in adults. Metastasis develops in almost half of the patients and over 90% of the metastases are in the liver. With the advances in molecular targeting therapy for melanoma, a proper metastasis ...
    Sensitive Estimation of Flavor Preferences in STFP Using Cumulative Time Profiles
    Authors:  Aditya Singh and J. Balaji, date: 11/05/2017, view: 215, Q&A: 0
    Social transmission of food preference (STFP) is observed among rodents between a demonstrator and a naïve hungry observer. During social interaction, hungry observer receives information about safety of the food consumed by the demonstrator. This ...
    Isolation of Primary Human Skeletal Muscle Cells
    Authors:  Janelle M. Spinazzola and Emanuela Gussoni, date: 11/05/2017, view: 177, Q&A: 0
    [Abstract] Primary myoblast culture is a valuable tool in research of muscle disease, pathophysiology, and pharmacology. This protocol describes techniques for dissociation of cells from human skeletal muscle biopsies and enrichment for a highly myogenic population by fluorescence-activated cell sorting (FACS). We also describe methods for assessing ...
    In vitro NLK Kinase Assay
    Authors:  Sungho Moon, Jiyoung Kim and Eek‐hoon Jho, date: 11/05/2017, view: 187, Q&A: 0
    [Abstract] This protocol provides step by step instructions to perform an in vitro kinase assay for nemo-like kinase. In addition, this protocol also describes an efficient method using mild lysis buffer for expression and purification of Glutathione S-transferase (GST) fusion proteins.
    Assessment of Aversion of Acute Pain Stimulus through Conditioned Place Aversion
    [Abstract] Pain is a complex experience. The aversive component of pain has been assessed through conditioned place aversion in rodents. However, this behavioral test does not allow the evaluation of the aversion of an acute pain stimulus. In Zhang et al. (2017), we provide an updated version of a Conditioned Place Aversion paradigm to address this ...
    Histochemical Staining of Collagen and Identification of Its Subtypes by Picrosirius Red Dye in Mouse Reproductive Tissues
    [Abstract] Collagen is one of the foremost components of tissue extracellular matrix (ECM). It provides strength, elasticity and architecture to the tissue enabling it to bear the wear and tear from external factors like physical stress as well as internal stress factors like inflammation or other pathological conditions. During normal pregnancy or pregnancy ...
    Isolation, Culture and Differentiation of Adult Hippocampal Precursor Cells
    Authors:  Stefanie N. Bernas, Odette Leiter, Tara L. Walker and Gerd Kempermann, date: 11/05/2017, view: 370, Q&A: 0
    [Abstract] There are two neurogenic niches in the adult mammalian brain: the subventricular zone of the lateral ventricle and the subgranular zone of the hippocampal dentate gyrus. Cells from these areas can be isolated and maintained in vitro, using two different culture systems to assess their potential regarding proliferation and differentiation ...
    Monitoring the Targeting of Cathepsin D to the Lysosome by Metabolic Labeling and Pulse-chase Analysis
    Authors:  Lucas A. Tavares and Luis L. P. daSilva, date: 11/05/2017, view: 220, Q&A: 0
    [Abstract] Mannose 6-phosphate receptors function can be studied in living cells by investigating alterations in processing and secretion of their ligand Cathepsin D. The assay described here is well established in the literature and comprises the metabolic labeling of newly synthesized proteins with [35S] methionine-cysteine in HeLa cells to ...
    Proximal Ligation Assay (PLA) on Lung Tissue and Cultured Macrophages to Demonstrate Protein-protein Interaction
    Authors:  Roberto Mendez and Santanu Banerjee, date: 11/05/2017, view: 182, Q&A: 0
    [Abstract] In this protocol, we describe proximal ligation assay (PLA), an antibody-based detection method for protein-protein interaction. This method relies on specific binding of individual primary antibodies to the two putative interacting proteins. The primary antibodies need to have different hosts. The secondary antibodies against the two hosts have ...
    Xenograft Mouse Model of Human Uveal Melanoma
    Authors:  Yao Chen, Xiao Liu, Ling Gao and Yongqing Liu, date: 11/05/2017, view: 161, Q&A: 0
    [Abstract] Uveal melanoma (UM) is a malignant intraocular tumor in adults. Metastasis develops in almost half of the patients and over 90% of the metastases are in the liver. With the advances in molecular targeting therapy for melanoma, a proper metastasis animal model is of increasing importance for testing the accuracy and effectiveness of systemic ...
    Sensitive Estimation of Flavor Preferences in STFP Using Cumulative Time Profiles
    Authors:  Aditya Singh and J. Balaji, date: 11/05/2017, view: 215, Q&A: 0
    [Abstract] Social transmission of food preference (STFP) is observed among rodents between a demonstrator and a naïve hungry observer. During social interaction, hungry observer receives information about safety of the food consumed by the demonstrator. This task has been implemented to develop a single trial non-aversive learning task in order to test ...
    Cytosolic and Nuclear Delivery of CRISPR/Cas9-ribonucleoprotein for Gene Editing Using Arginine Functionalized Gold Nanoparticles
    Authors:  Rubul Mout and Vincent M. Rotello, date: 10/20/2017, view: 683, Q&A: 0
    [Abstract] In this protocol, engineered Cas9-ribonucleoprotein (Cas9 protein and sgRNA, together called Cas9-RNP) and gold nanoparticles are used to make nanoassemblies that are employed to deliver Cas9-RNP into cell cytoplasm and nucleus. Cas9 protein is engineered with an N-terminus glutamic acid tag (E-tag or En, where n = the number of glutamic acid in ...