Mammalia

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    Protocols in Current Issue
    Simple Protocol for Distinguishing Drug-induced Effects on Spatial Memory Acquisition, Consolidation and Retrieval in Mice Using the Morris Water Maze
    Authors:  Ariel K. Frame, Asad Lone, Richard A. Harris and Robert C. Cumming, date: 09/20/2019, view: 52, Q&A: 0
    The Morris water maze (MWM) is one of the most commonly used tests for assessing spatial learning and memory in mice. While the MWM is highly amenable to testing the effects of memory modifying drugs, most studies do not consider the timing or ...
    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: 41, Q&A: 0
    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 ...
    Conjugation of Fab’ Fragments with Fluorescent Dyes for Single-molecule Tracking on Live Cells
    Authors:  I-Ting Teng, Xiangning Bu and Inhee Chung, date: 09/20/2019, view: 50, Q&A: 0
    Our understanding of the regulation and functions of cell-surface proteins has progressed rapidly with the advent of advanced optical imaging techniques. In particular, single-molecule tracking (SMT) using bright fluorophores conjugated to ...
    Immunohistochemical Staining of CD8α in Diabetic Mouse Kidney
    Authors:  Ming-Zhi Zhang and Raymond C. Harris, date: 09/20/2019, view: 46, Q&A: 0
    Immune cell infiltration, particularly cytotoxic CD8α lymphocyte infiltration, plays an important role in development of diabetic nephropathy. Although CD8α infiltration can be evaluated by its production of cytokines, its localization in the kidney ...
    SIRF: A Single-cell Assay for in situ Protein Interaction with Nascent DNA Replication Forks
    Authors:  Sunetra Roy and Katharina Schlacher, date: 09/20/2019, view: 52, Q&A: 0
    The duplication of DNA is a fundamental process that is required for the transfer of the genetic information from parent to daughter cells. Aberrant DNA replication processes are associated with diverse disease phenotypes, including developmental ...
    Subcellular Fractionation of Hela Cells for Lysosome Enrichment Using a Continuous Percoll-density Gradient
    The enrichment of lysosomes is a useful way to study their structure and function. These dynamic vesicles can be enriched from cell cultures in a variety of ways including immunoprecipitation and fluorescence-activated organelle sorting. These ...
    Supported Cell Membrane Sheets to Monitor Protein Assembly
    Authors:  Simon Erlendsson, Thor Seneca Thorsen and Kenneth Lindegaard Madsen, date: 09/20/2019, view: 48, Q&A: 0
    Studying protein-protein and protein-lipid interactions in their native environment is highly desirable, yet, the heterogeneity and complexity of cellular systems limits the repertoire of experimental methods available. In cells, interactions are ...
    Characterization of Biological Motion Using Motion Sensing Superpixels
    Precise spatiotemporal regulation is the foundation for the healthy development and maintenance of living organisms. All cells must correctly execute their function in the right place at the right time. Cellular motion is thus an important dynamic ...
    Sciatic Nerve Cut and Repair Using Fibrin Glue in Adult Mice
    Authors:  Erica T. Akhter, Travis M. Rotterman, Arthur W. English and Francisco J. Alvarez, date: 09/20/2019, view: 47, Q&A: 0
    Peripheral nerve injury (PNI) is an excellent model for studying neural responses to injury and elucidating the mechanisms that can facilitate axon regeneration. As such, several animal models have been employed to study regenerative mechanisms ...
    Explant Culture of the Embryonic Mouse Spinal Cord and Gene Transfer by ex vivo Electroporation
    Developing axons change responsiveness to guidance cues during the journey to synapse with target cells. Axon crossing at the ventral midline serves as a model for studying how axons accomplish such a switch in their response. Although primary ...
    A Cell Culture Model that Mimics Physiological Tissue Oxygenation Using Oxygen-permeable Membranes
    Authors:  Chloe-Anne Martinez, Peter A. Cistulli and Kristina M. Cook, date: 09/20/2019, view: 78, Q&A: 0
    Dissolved oxygen and its availability to cells in culture is an overlooked variable which can have significant consequences on experimental research outcomes, including reproducibility. Oxygen sensing pathways play key roles in cell growth and ...
    Simple Protocol for Distinguishing Drug-induced Effects on Spatial Memory Acquisition, Consolidation and Retrieval in Mice Using the Morris Water Maze
    Authors:  Ariel K. Frame, Asad Lone, Richard A. Harris and Robert C. Cumming, date: 09/20/2019, view: 52, Q&A: 0
    [Abstract] The Morris water maze (MWM) is one of the most commonly used tests for assessing spatial learning and memory in mice. While the MWM is highly amenable to testing the effects of memory modifying drugs, most studies do not consider the timing or duration of drug exposure when conducting the MWM assay; factors that can strongly influence the effect ...
    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: 41, 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.
    Conjugation of Fab’ Fragments with Fluorescent Dyes for Single-molecule Tracking on Live Cells
    Authors:  I-Ting Teng, Xiangning Bu and Inhee Chung, date: 09/20/2019, view: 50, Q&A: 0
    [Abstract] Our understanding of the regulation and functions of cell-surface proteins has progressed rapidly with the advent of advanced optical imaging techniques. In particular, single-molecule tracking (SMT) using bright fluorophores conjugated to antibodies and wide-field microscopy methods such as total internal reflection fluorescence microscopy have ...
    Immunohistochemical Staining of CD8α in Diabetic Mouse Kidney
    Authors:  Ming-Zhi Zhang and Raymond C. Harris, date: 09/20/2019, view: 46, Q&A: 0
    [Abstract] Immune cell infiltration, particularly cytotoxic CD8α lymphocyte infiltration, plays an important role in development of diabetic nephropathy. Although CD8α infiltration can be evaluated by its production of cytokines, its localization in the kidney is of particular importance. The current protocol describes CD8α immunostaining using a Vectastain ...
    SIRF: A Single-cell Assay for in situ Protein Interaction with Nascent DNA Replication Forks
    Authors:  Sunetra Roy and Katharina Schlacher, date: 09/20/2019, view: 52, Q&A: 0
    [Abstract] The duplication of DNA is a fundamental process that is required for the transfer of the genetic information from parent to daughter cells. Aberrant DNA replication processes are associated with diverse disease phenotypes, including developmental defects, ageing disorders, blood disorders such as Fanconi Anemia, increased inflammation and cancer. ...
    Subcellular Fractionation of Hela Cells for Lysosome Enrichment Using a Continuous Percoll-density Gradient
    [Abstract] The enrichment of lysosomes is a useful way to study their structure and function. These dynamic vesicles can be enriched from cell cultures in a variety of ways including immunoprecipitation and fluorescence-activated organelle sorting. These methods are extremely precise but often require the transfection and expression of an affinity or ...
    Supported Cell Membrane Sheets to Monitor Protein Assembly
    Authors:  Simon Erlendsson, Thor Seneca Thorsen and Kenneth Lindegaard Madsen, date: 09/20/2019, view: 48, Q&A: 0
    [Abstract] Studying protein-protein and protein-lipid interactions in their native environment is highly desirable, yet, the heterogeneity and complexity of cellular systems limits the repertoire of experimental methods available. In cells, interactions are often taking place in confined microenvironments where factors such as avidity, hindered diffusion, ...
    Characterization of Biological Motion Using Motion Sensing Superpixels
    [Abstract] Precise spatiotemporal regulation is the foundation for the healthy development and maintenance of living organisms. All cells must correctly execute their function in the right place at the right time. Cellular motion is thus an important dynamic readout of signaling in key disease-relevant molecular pathways. However despite the rapid ...
    Sciatic Nerve Cut and Repair Using Fibrin Glue in Adult Mice
    Authors:  Erica T. Akhter, Travis M. Rotterman, Arthur W. English and Francisco J. Alvarez, date: 09/20/2019, view: 47, Q&A: 0
    [Abstract] Peripheral nerve injury (PNI) is an excellent model for studying neural responses to injury and elucidating the mechanisms that can facilitate axon regeneration. As such, several animal models have been employed to study regenerative mechanisms after PNI, including Aplysia, zebrafish, rabbits, cats and rodents. This protocol describes how to ...
    Explant Culture of the Embryonic Mouse Spinal Cord and Gene Transfer by ex vivo Electroporation
    [Abstract] Developing axons change responsiveness to guidance cues during the journey to synapse with target cells. Axon crossing at the ventral midline serves as a model for studying how axons accomplish such a switch in their response. Although primary neuron culture has been a versatile technique for elucidating various developmental mechanisms, many in ...



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