Protocols in Current Issue
    Measurement of TLR4 and CD14 Receptor Endocytosis Using Flow Cytometry
    Authors:  Michael S. Schappe and Bimal N. Desai, date: 07/20/2018, view: 494, Q&A: 0
    [Abstract] After recognizing extracellular bacterial lipopolysaccharide (LPS), the toll-like receptor 4 (TLR4)-CD14 signaling complex initiates two distinct signaling pathways–one from the plasma membrane and the other from the signaling endosomes (Kagan et al., 2008). Understanding the early stages of TLR4 signal transduction therefore requires a ...
    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: 582, 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 ...
    Immunohistochemical Identification of Human Skeletal Muscle Macrophages
    [Abstract] Macrophages have well-characterized roles in skeletal muscle repair and regeneration. Relatively little is known regarding the role of resident macrophages in skeletal muscle homeostasis, extracellular matrix remodeling, growth, metabolism and adaptation to various stimuli including exercise and training. Despite speculation into macrophage ...
    Visualization of RNA at the Single Cell Level by Fluorescent in situ Hybridization Coupled to Flow Cytometry
    Authors:  Alice Coillard and Elodie Segura, date: 06/20/2018, view: 642, Q&A: 0
    [Abstract] The protocol described here has been developed to detect RNA at the single cell level. Fluorescent probes hybridize to target RNAs and are detected by flow cytometry after multiple amplification steps. Different types of RNA can be detected such as mRNA, long noncoding RNA, viral RNA or telomere RNA and up to 4 different target probes can be used ...
    High Dimensional Functionomic Analysis of Human Hematopoietic Stem and Progenitor Cells at a Single Cell Level
    Authors:  Thomas Luh, Kimberly Lucero, Wenji Ma, Jaeyop Lee, Yu Jerry Zhou, Yufeng Shen and Kang Liu, date: 05/20/2018, view: 838, Q&A: 0
    [Abstract] The ability to conduct investigation of cellular transcription, signaling, and function at the single-cell level has opened opportunities to examine heterogeneous populations at unprecedented resolutions. Although methods have been developed to evaluate high-dimensional transcriptomic and proteomic data (relating to cellular mRNA and protein), ...
    Purification of Total RNA from DSS-treated Murine Tissue via Lithium Chloride Precipitation
    Authors:  Emilie Viennois, Anika Tahsin and Didier Merlin, date: 05/05/2018, view: 816, Q&A: 0
    [Abstract] We have developed a protocol to purify RNA from DSS (Dextran Sulfate Sodium)-treated mouse tissues. This method, which prevents downstream inhibition of q-RT-PCR observed in DSS-treated tissues, relies on successive precipitations with lithium chloride.
    Nab Escaping AAV Mutants Isolated from Mouse Muscles
    Authors:  Zheng Chai, R. Jude Samulski and Chengwen Li, date: 05/05/2018, view: 814, Q&A: 0
    [Abstract] Neutralizing antibodies (Nabs) are a major challenge in clinical trials of adeno-associated virus (AAV) vector gene therapy, because Nabs are able to inhibit AAV transduction in patients. We have successfully isolated several novel Nab-escaped AAV chimeric capsids in mice by administrating a mixture of AAV shuffled library and patient serum. These ...
    3D Co-culture System of Tumor-associated Macrophages and Ovarian Cancer Cells
    Authors:  Lingli Long, Mingzhu Yin and Wang Min, date: 04/20/2018, view: 1280, Q&A: 0
    [Abstract] Ovarian cancer is fairly unique in that ovarian carcinoma cells can detach and spread directly through peritoneal cavity. It has been unclear, however, how detached cancer cells survive in the peritoneum and form spheroid structure. We have recently reported that there is a strong correlation between Tumor-associated macrophages (TAMs)-associated ...
    Adhesion of Enteroaggregative E. coli Strains to HEK293 Cells
    Authors:  Jorge Luis Ayala-Lujan and Fernando Ruiz-Perez, date: 04/20/2018, view: 825, Q&A: 0
    [Abstract] Enteroaggregative Escherichia coli (EAEC) is a recognized cause of acute diarrhea among both children and adults worldwide. EAEC strains are characterized by the presence of aggregative adherence fimbriae (AAF), which play a key role in pathogenesis by mediating attachment to the intestinal mucosa and by triggering host inflammatory ...
    Imaging Cytokine Concentration Fields Using PlaneView Imaging Devices
    Authors:  Alon Oyler-Yaniv and Oleg Krichevsky, date: 04/05/2018, view: 918, 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 ...