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    Protocols in Current Issue
    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: 34, Q&A: 0
    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 ...
    Protocol for Notch-ligand Binding Assays Using Dynabeads
    Authors:  Shogo Sawaguchi, Mitsutaka Ogawa and Tetsuya Okajima, date: 10/20/2017, view: 25, Q&A: 0
    This protocol describes how to measure interaction between Notch receptors and their ligands by cell-based assay using Dynabeads. We have used the protocol to determine binding capacity between Notch1-transfected HEK293T cells and ligand-coated ...
    Labeling Aversive Memory Trace in Mouse Using a Doxycycline-inducible Expression System
    Authors:  Jianyang Du and Erin E. Koffman, date: 10/20/2017, view: 32, Q&A: 0
    A memory trace, also known as a memory engram, is theorized to be a mechanism for physical memory storage in the brain (Silva et al., 2009; Josselyn, 2010) and memory trace is associated with a specific population of neurons (Liu et al. ...
    Isolation and Purification of Schwann Cells from Spinal Nerves of Neonatal Rat
    Authors:  Jinkun Wen, Dandan Tan, Lixia Li and Jiasong Guo, date: 10/20/2017, view: 38, Q&A: 0
    Primary cultured Schwann cells (SCs) are widely used in the investigation of the biology of SC and are important seed cells for neural tissue engineering. Here, we describe a novel protocol for harvesting primary cultured SCs from neonatal ...
    In vitro Co-culture of Mesenchymal Stem Cells and Endothelial Colony Forming Cells
    Authors:  Abbas Shafiee and Kiarash Khosrotehrani, date: 10/20/2017, view: 61, Q&A: 0
    The discovery of endothelial colony forming cells (ECFCs) with robust self-renewal and de novo vessel formation potentials suggests that ECFCs can be an excellent cell source for cardiovascular diseases treatment through improving neovascularization ...
    NP-40 Fractionation and Nucleic Acid Extraction in Mammalian Cells
    Authors:  Alvaro E. Galvis, Hugh E. Fisher and David Camerini, date: 10/20/2017, view: 92, Q&A: 0
    This technique allows for efficient, highly purified cytoplasmic and nuclear-associated compartment fractionation utilizing NP-40 detergent in mammalian cells. The nuclear membrane is not disturbed during the fractionation thus leaving all nuclear ...
    Touchscreen-based Visual Discrimination and Reversal Tasks for Mice to Test Cognitive Flexibility
    Authors:  Karly M. Turner, Christopher G. Simpson and Thomas H. J. Burne, date: 10/20/2017, view: 17, Q&A: 0
    Reversal learning can be used to examine deficits in cognitive flexibility, which have been linked to a number of neuropsychiatric disorders including schizophrenia and addiction. However, methods of examining reversal learning have varied ...
    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: 34, 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 ...
    Protocol for Notch-ligand Binding Assays Using Dynabeads
    Authors:  Shogo Sawaguchi, Mitsutaka Ogawa and Tetsuya Okajima, date: 10/20/2017, view: 25, Q&A: 0
    [Abstract] This protocol describes how to measure interaction between Notch receptors and their ligands by cell-based assay using Dynabeads. We have used the protocol to determine binding capacity between Notch1-transfected HEK293T cells and ligand-coated Dynabeads. Expression of Eogt in Notch1-expressing cells promoted binding toward DLL4-coated ...
    Labeling Aversive Memory Trace in Mouse Using a Doxycycline-inducible Expression System
    Authors:  Jianyang Du and Erin E. Koffman, date: 10/20/2017, view: 32, Q&A: 0
    [Abstract] A memory trace, also known as a memory engram, is theorized to be a mechanism for physical memory storage in the brain (Silva et al., 2009; Josselyn, 2010) and memory trace is associated with a specific population of neurons (Liu et al., 2012; Ramirez et al., 2013). Labeling and stimulating those neurons will activate ...
    Isolation and Purification of Schwann Cells from Spinal Nerves of Neonatal Rat
    Authors:  Jinkun Wen, Dandan Tan, Lixia Li and Jiasong Guo, date: 10/20/2017, view: 38, Q&A: 0
    [Abstract] Primary cultured Schwann cells (SCs) are widely used in the investigation of the biology of SC and are important seed cells for neural tissue engineering. Here, we describe a novel protocol for harvesting primary cultured SCs from neonatal Sprague-Dawley (SD) rats. In the present protocol, dissociated SCs are isolated from the spinal nerves of ...
    In vitro Co-culture of Mesenchymal Stem Cells and Endothelial Colony Forming Cells
    Authors:  Abbas Shafiee and Kiarash Khosrotehrani, date: 10/20/2017, view: 61, Q&A: 0
    [Abstract] The discovery of endothelial colony forming cells (ECFCs) with robust self-renewal and de novo vessel formation potentials suggests that ECFCs can be an excellent cell source for cardiovascular diseases treatment through improving neovascularization in the ischemic tissues. However, their engraftment after transplantation resulted to be low. ...
    NP-40 Fractionation and Nucleic Acid Extraction in Mammalian Cells
    Authors:  Alvaro E. Galvis, Hugh E. Fisher and David Camerini, date: 10/20/2017, view: 92, Q&A: 0
    [Abstract] This technique allows for efficient, highly purified cytoplasmic and nuclear-associated compartment fractionation utilizing NP-40 detergent in mammalian cells. The nuclear membrane is not disturbed during the fractionation thus leaving all nuclear and perinuclear associated components in the nuclear fraction. This protocol has been modified from ...
    Touchscreen-based Visual Discrimination and Reversal Tasks for Mice to Test Cognitive Flexibility
    Authors:  Karly M. Turner, Christopher G. Simpson and Thomas H. J. Burne, date: 10/20/2017, view: 17, Q&A: 0
    [Abstract] Reversal learning can be used to examine deficits in cognitive flexibility, which have been linked to a number of neuropsychiatric disorders including schizophrenia and addiction. However, methods of examining reversal learning have varied substantially between species. Touchscreen technology has allowed researchers to explore cognitive deficits ...
    Protein Expression and Purification of the Hsp90-Cdc37-Cdk4 Kinase Complex from Saccharomyces cerevisiae
    Authors:  Kliment A. Verba and David A. Agard, date: 10/05/2017, view: 356, Q&A: 0
    [Abstract] Interactions between Hsp90, its co-chaperone Cdc37 and kinases have been biochemically studied for over three decades and have been shown to be functionally important in organisms from yeast to humans. However, formation of a stable complex for structural studies has been elusive. In this protocol we describe expression and purification of ...
    DQ-Red BSA Trafficking Assay in Cultured Cells to Assess Cargo Delivery to Lysosomes
    Authors:  Rituraj Marwaha and Mahak Sharma, date: 10/05/2017, view: 774, Q&A: 0
    [Abstract] Lysosomes are the terminal end of the endocytic pathway having acidic environment required for active hydrolases that degrade the cargo delivered to these compartments. This process of cargo delivery and degradation by endo-lysosomes is a tightly regulated process and important for maintaining cellular homeostasis. Cargos like EGF (Epidermal ...
    Accurate, Streamlined Analysis of mRNA Translation by Sucrose Gradient Fractionation
    Authors:  Soufiane Aboulhouda, Rachael Di Santo, Gabriel Therizols and David Weinberg, date: 10/05/2017, view: 276, Q&A: 0
    [Abstract] The efficiency with which proteins are produced from mRNA molecules can vary widely across transcripts, cell types, and cellular states. Methods that accurately assay the translational efficiency of mRNAs are critical to gaining a mechanistic understanding of post-transcriptional gene regulation. One way to measure translational efficiency is to ...