Protocols in Current Issue
    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 ...
    Induced Germinal Center B Cell Culture System
    Authors:  Kei Haniuda and Daisuke Kitamura, date: 02/20/2019, view: 1094, Q&A: 0
    [Abstract] The germinal center (GC) is the site where B cells undergo clonal expansion, affinity-based selection, and differentiation into memory B cells or plasma cells. It has been difficult to elucidate regulatory mechanisms for the dynamic GC B cell maturation and differentiation, partly because experimental manipulation of GC B cells in vivo ...
    Isolation of Chromatin-bound Proteins from Subcellular Fractions for Biochemical Analysis
    Author:  Sébastien Gillotin, date: 10/05/2018, view: 2543, Q&A: 1
    [Abstract] Shuttling of proteins between different cellular compartments controls their proteostasis and can contribute in some cases to regulate their activity. Biochemical analysis of chromatin-bound proteins, such as transcription factors, is often difficult because of their low yield and due to the interference from nucleic acids. This protocol describes ...
    Quantification of Extracellular Double-stranded RNA Uptake and Subcellular Localization Using Flow Cytometry and Confocal Microscopy
    Authors:  Tan A Nguyen, Lachlan Whitehead and Ken C Pang, date: 06/20/2018, view: 1870, Q&A: 0
    [Abstract] Double-stranded RNA is a potent pathogen-associated molecular pattern (PAMP) produced as a by-product of viral replication and a well-known hallmark of viral infection. Viral dsRNAs can be released from infected cells into the extracellular space and internalized by neighboring cells via endocytosis. Mammals possess multiple pattern recognition ...
    Quantifying Podocytes and Parietal Epithelial Cells in Human Urine Using Liquid-based Cytology and WT1 Immunoenzyme Staining
    [Abstract] In glomerular disease, podocytes and parietal epithelial cells (PECs) are shed in the urine. Therefore, urinary podocytes and PECs are noninvasive biomarkers of glomerular disease. The purpose of this protocol is to employ immunocytochemistry to detect podocytes and PECs, using the WT1 antibody on liquid-based cytology slides.
    Imaging Cytokine Concentration Fields Using PlaneView Imaging Devices
    Authors:  Alon Oyler-Yaniv and Oleg Krichevsky, date: 04/05/2018, view: 2153, Q&A: 0
    [Abstract] We describe here a method to visualize concentration fields of cytokines around cytokine-secreting cells. The main challenge is that physiological cytokine concentrations can be very low, in the pico-molar range. Since it is currently impossible to measure such concentrations directly, we rely on cell’s response to the cytokines–the ...
    Quantitative Live-cell Reporter Assay for Noncanonical Wnt Activity
    Authors:  Edith P. Karuna, Michael W. Susman and Hsin-Yi Henry Ho, date: 03/20/2018, view: 2931, Q&A: 0
    [Abstract] Noncanonical Wnt signaling functions independently of the β-catenin pathway to control diverse developmental processes, and dysfunction of the pathway contributes to a number of human pathological conditions, including birth defects and metastatic cancer. Progress in the field, however, has been hampered by the scarcity of functional assays for ...
    Flow Cytometric Quantification of Fatty Acid Uptake by Mycobacterium tuberculosis in Macrophages
    Authors:  Evgeniya V. Nazarova, Maria Podinovskaia, David G. Russell and Brian C. VanderVen, date: 02/20/2018, view: 3072, Q&A: 0
    [Abstract] Mycobacterium tuberculosis (Mtb) has evolved to assimilate fatty acids from its host. However, until recently, there was no reliable way to quantify fatty acid uptake by the bacteria during host cell infection. Here we describe a new method to quantify fatty acid uptake by intracellular bacilli. We infect macrophages with Mtb ...
    Lentiviral Knockdown of Transcription Factor STAT1 in Peromyscus leucopus to Assess Its Role in the Restriction of Tick-borne Flaviviruses
    Authors:  Adaeze O. Izuogu and R. Travis Taylor, date: 12/05/2017, view: 3740, Q&A: 0
    [Abstract] Cellular infection with tick-borne flaviviruses (TBFVs) results in activation of the interferon (IFN) signaling pathway and subsequent upregulation of numerous genes termed IFN stimulated genes (ISGs) (Schoggins et al., 2011). Many ISGs function to prevent virus pathogenesis by acting in a broad or specific manner through protein-protein ...
    Myeloid Progenitor Transformation Assay
    Authors:  Hiroshi Okuda and Akihiko Yokoyama, date: 12/05/2017, view: 2756, Q&A: 0
    [Abstract] Numerous oncogenes have been identified to cause leukemia. For example, chromosomal translocation generates various fusion genes of the mixed-lineage leukemia (MLL) gene and a partner gene in leukemia, whose gene products transform primary myeloid progenitors into an immortalized state. To characterize the transforming ability of leukemic ...

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