Mammalia

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    Protocols in Current Issue
    TZA, a Sensitive Reporter Cell-based Assay to Accurately and Rapidly Quantify Inducible, Replication-competent Latent HIV-1 from Resting CD4+ T Cells
    Authors:  Anwesha Sanyal, Vatsala S. Rangachar and Phalguni Gupta, date: 05/20/2019, view: 221, Q&A: 0
    The latent HIV-1 viral reservoir in resting CD4+ (rCD4+) T cells represents a major barrier to an HIV-1 cure. There is an ongoing effort to identify therapeutic approaches that will eliminate or reduce the size of this ...
    Protocol to Quantitatively Assess the Structural Integrity of Perineuronal Nets ex vivo
    Authors:  Bhanu P. Tewari and Harald Sontheimer, date: 05/20/2019, view: 210, Q&A: 0
    Perineuronal nets (PNNs) are extracellular matrix assemblies of highly negatively charged proteoglycans that wrap around fast-spiking parvalbumin (PV) expressing interneurons in the cerebral cortex. PNNs play important roles in neuronal plasticity ...
    Simultaneous Fluorescent Recordings of Extracellular ATP and Intracellular Calcium in Mammalian Cells
    Authors:  Nicholas Mikolajewicz and Svetlana V. Komarova, date: 05/20/2019, view: 444, Q&A: 0
    Extracellular ATP is a potent signaling molecule that stimulates intracellular calcium responses through purinergic (P2) receptors in mammalian cells. While extracellular ATP and intracellular calcium can be measured separately, simultaneous ...
    A Flow Cytometric Method to Determine Transfection Efficiency
    Mammalian cell transfection is a powerful technique commonly used in molecular biology to express exogenous DNA or RNA in cells and study gene and protein function. Although several transfection strategies have been developed, there is a wide ...
    TZA, a Sensitive Reporter Cell-based Assay to Accurately and Rapidly Quantify Inducible, Replication-competent Latent HIV-1 from Resting CD4+ T Cells
    Authors:  Anwesha Sanyal, Vatsala S. Rangachar and Phalguni Gupta, date: 05/20/2019, view: 221, Q&A: 0
    [Abstract] The latent HIV-1 viral reservoir in resting CD4+ (rCD4+) T cells represents a major barrier to an HIV-1 cure. There is an ongoing effort to identify therapeutic approaches that will eliminate or reduce the size of this reservoir. However, clinical investigators lack an assay to determine whether or not a decrease in the ...
    Protocol to Quantitatively Assess the Structural Integrity of Perineuronal Nets ex vivo
    Authors:  Bhanu P. Tewari and Harald Sontheimer, date: 05/20/2019, view: 210, Q&A: 0
    [Abstract] Perineuronal nets (PNNs) are extracellular matrix assemblies of highly negatively charged proteoglycans that wrap around fast-spiking parvalbumin (PV) expressing interneurons in the cerebral cortex. PNNs play important roles in neuronal plasticity and modulate biophysical properties of the enclosed interneurons. Various central nervous system ...
    Simultaneous Fluorescent Recordings of Extracellular ATP and Intracellular Calcium in Mammalian Cells
    Authors:  Nicholas Mikolajewicz and Svetlana V. Komarova, date: 05/20/2019, view: 444, Q&A: 0
    [Abstract] Extracellular ATP is a potent signaling molecule that stimulates intracellular calcium responses through purinergic (P2) receptors in mammalian cells. While extracellular ATP and intracellular calcium can be measured separately, simultaneous monitoring can offer additional insights into P2 receptor physiology. This protocol takes advantage of the ...
    A Flow Cytometric Method to Determine Transfection Efficiency
    [Abstract] Mammalian cell transfection is a powerful technique commonly used in molecular biology to express exogenous DNA or RNA in cells and study gene and protein function. Although several transfection strategies have been developed, there is a wide variation with regards to transfection efficiency, cell toxicity and reproducibility. Thus, a sensitive ...
    3D Co-culture System of Mouse Prostatic Wild-type Fibroblasts with Human Prostate Cancer Epithelial Cells
    Authors:  Veronica R. Placencio-Hickok, Manabu Kato and Neil A. Bhowmick, date: 05/05/2019, view: 433, Q&A: 0
    [Abstract] Heterogeneous prostatic carcinoma-associated fibroblasts (CAF) contribute to tumor progression. This was established using transgenic mouse models. Paracrine interactions between fibroblasts and epithelial cells were further interrogated using isolated 2D cell culture systems, but 3D culture systems currently being developed can better mimic ...
    Improved HTGTS for CRISPR/Cas9 Off-target Detection
    Authors:  Jianhang Yin, Mengzhu Liu, Yang Liu and Jiazhi Hu, date: 05/05/2019, view: 271, Q&A: 0
    [Abstract] Precise genome editing is essential for scientific research and clinical application. At present, linear amplification-mediated high-throughput genome-wide translocation sequencing (LAM-HTGTS) is one of most effective methods to evaluate the off-target activity of CRISPR-Cas9, which is based on chromosomal translocation and employs a “bait” DNA ...
    Triple Fluorescence Anisotropy Reporter Imaging in Living Cells
    Authors:  Brian Ross, Shenq Huey Wong, Nicole E. Snell, Jin Zhang and M. A. Rizzo, date: 05/05/2019, view: 370, Q&A: 0
    [Abstract] FRET-based genetically encoded biosensors incorporate two fluorescent proteins into their design to enable ratiometric biosensing of signaling activities in live cells. While emission ratios are generally useful for quantitative studies, they leave little room in the optical spectrum for additional sensors and optogenetic tools. Homotransfer-based ...
    Inducing Alcohol Dependence in Rats Using Chronic Intermittent Exposure to Alcohol Vapor
    Authors:  Elizabeth M. Avegno and Nicholas W. Gilpin, date: 05/05/2019, view: 236, Q&A: 0
    [Abstract] Alcohol use disorder (AUD) is a significant public health and economic burden and is often characterized by repeated bouts of alcohol intoxication and withdrawal. Virtually all organ systems are impacted by chronic alcohol exposure. These effects can be investigated using the rat as a model organism; however, rats typically will not ...
    Separation of Natural Collagen Crosslinks Using Buffer and Ion-pairing Agent Free Solvents on Silica Hydride Column for Mass Spectrometry Detection
    Authors:  Rafea Naffa and Joseph Pesek, date: 05/05/2019, view: 264, Q&A: 0
    [Abstract] In this protocol we describe the separation of collagen crosslinks in biological tissues and samples including skin, tendon, cartilage, bone and urine. The existing methods use either cation exchange chromatography followed by post-column derivatization with ninhydrin or reverse phase chromatography with mass spectrometry detection. The cation ...
    Optimized Oxidative Stress Protocols for Low-microliter Volumes of Mammalian Plasma
    Author:  Evan A. Langille, date: 05/05/2019, view: 323, Q&A: 0
    [Abstract] Small blood volumes commonly obtained from small mammals during field studies are only sufficient for a single biochemical assay. In this study, we used blood collected from a population of wild eastern chipmunks (Tamias striatus) and developed modified methods to improve analytical selectivity and sensitivity required for measuring ...



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