Mammalia

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    Protocols in Current Issue
    In situ Hybridization of miRNAs in Human Embryonic Kidney and Human Pluripotent Stem Cell-derived Kidney Organoids
    Authors:  Filipa M. Lopes, Susan J. Kimber and Ioannis Bantounas, date: 09/05/2021, view: 1179, Q&A: 0
    [Abstract]

    MicroRNAs are small RNAs that negatively regulate gene expression and play an important role in fine-tuning molecular pathways during development. There is increasing interest in studying their function in the kidney, but the majority of studies to date use kidney cell lines and assess the total amounts of miRNAs of interest either by qPCR or by

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    Method for Rapid Enzymatic Cleaning for Reuse of Patch Clamp Pipettes: Increasing Throughput by Eliminating Manual Pipette Replacement between Patch Clamp Attempts
    [Abstract]

    The whole-cell patch-clamp method is a gold standard for single-cell analysis of electrical activity, cellular morphology, and gene expression. Prior to our discovery that patch-clamp pipettes could be cleaned and reused, experimental throughput and automation were limited by the need to replace pipettes manually after each experiment. This

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    Carboxyfluorescein Dye Uptake to Measure Connexin-mediated Hemichannel Activity in Cultured Cells
    [Abstract]

    Connexins are membrane bound proteins that facilitate direct and local paracrine mediated cell-to-cell communication through their ability to oligomerise into hexameric hemichannels. When neighbouring channels align, they form gap-junctions that provide a direct route for information transfer between cells. In contrast to intact gap junctions,

    ...
    Double Labeling of PDGFR-β and α-SMA in Swine Models of Acute Kidney Injury to Detect Pericyte-to-Myofibroblast Transdifferentation as Early Marker of Fibrosis
    [Abstract] Growing evidences suggest that peritubular capillaries pericytes are the main source of scar-forming myofibroblasts during chronic kidney disease (CKD), as well as early phases of acute kidney injury (AKI). In a swine model of sepsis and I/R (Ischemia Reperfusion) injury-induced AKI we demonstrated that renal pericytes are able to ...
    Immunoprecipitation of Acetyl-lysine and Western Blotting of Long-chain acyl-CoA Dehydrogenases and Beta-hydroxyacyl-CoA Dehydrogenase in Palmitic Acid Treated Human Renal Tubular Epithelial Cells
    Authors:  Tingting Lv, Suwei Zhu, Yuan Ma, Hong Feng and Qiang Wan, date: 09/20/2020, view: 2893, Q&A: 0
    [Abstract] As one of the main energy metabolism organs, kidney has been proved to have high energy requirements and are more inclined to fatty acid metabolism as the main energy source. Long-chain acyl-CoA dehydrogenases (LCAD) and beta-hydroxyacyl-CoA dehydrogenase (beta-HAD), key enzymes involved in fatty acid oxidation, has been identified as the ...
    Assessing Gαq/15-signaling with IP-One: Single Plate Transfection and Assay Protocol for Cell-Based High-Throughput Assay
    [Abstract] Cell-based functional assays are an important part of compound screening and drug lead optimization, and they can also play a crucial role in the determination of the residues involved in ligand binding and signaling for a particular G-protein-coupled receptor. Conventional methods used for Gαq/15-coupled receptors rely on the use of ...
    FRET Reporter Assays for cAMP and Calcium in a 96-well Format Using Genetically Encoded Biosensors Expressed in Living Cells
    Authors:  Brandon T. Milliken, Robert P. Doyle, George G. Holz and Oleg G. Chepurny, date: 06/05/2020, view: 4028, Q&A: 0
    [Abstract] Stimulation of G protein-coupled receptors (GPCR) by hormones and neurotransmitters elicits cellular responses, many of which result from alterations in the concentrations of cytosolic cAMP and Ca2+. Here, we describe a microplate reader fluorescence resonance energy transfer (FRET) assay that uses the genetically encoded biosensors ...
    Using Imaging Flow Cytometry to Characterize Extracellular Vesicles Isolated from Cell Culture Media, Plasma or Urine
    Authors:  John R. Woollard, Amrutesh Puranik, Kyra L. Jordan and Lilach O. Lerman, date: 11/05/2019, view: 3012, Q&A: 0
    [Abstract] The ability to non-invasively detect specific damage to the kidney has been limited. Identification of extracellular vesicles released by cells, especially when under duress, might allow for monitoring and identification of specific cell types within the kidney that are stressed. We have adapted a previously published traditional flow cytometry ...
    Supported Cell Membrane Sheets to Monitor Protein Assembly
    Authors:  Simon Erlendsson, Thor Seneca Thorsen and Kenneth Lindegaard Madsen, date: 09/20/2019, view: 3803, 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, ...
    Determination of Cellular Uptake and Endocytic Pathways
    Authors:  Jing Gong, Hong-Xia Wang and Kam W. Leong, date: 02/20/2019, view: 7636, Q&A: 0
    [Abstract] Efficiency of drug and gene delivery via nonviral vehicles is contingent on proper cellular uptake and intracellular release. Further, various cargos, such as nucleases for gene editing or inhibitors for endosomal receptors, require transport to specific compartments of the cell. Hence, characterization of cellular uptake and endocytic pathways is ...



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