Cell Biology

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    Protocols in Current Issue
    A Flow Cytometry-Based Method for Analyzing DNA End Resection in G0- and G1-Phase Mammalian Cells
    Authors:  Bo-Ruei Chen, Jessica K. Tyler and Barry P. Sleckman, date: 05/20/2022, view: 978, Q&A: 0
    [Abstract]

    DNA double strand breaks (DSBs) constantly arise in cells during normal cellular processes or upon exposure to genotoxic agents, and are repaired mostly by homologous recombination (HR) and non-homologous end joining (NHEJ). One key determinant of DNA DSB repair pathway choice is the processing of broken DNA ends to generate single strand DNA

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    Flow Cytometry-based Measurement of Reactive Oxygen Species in Cyanobacteria
    Authors:  Soumila Mondal and Shailendra P. Singh, date: 05/20/2022, view: 858, Q&A: 0
    [Abstract]

    Cyanobacteria are Gram-negative oxygen-producing photosynthetic bacteria that are useful in the pharmaceutical and biofuel industries. Monitoring of oxidative stress under fluctuating environmental conditions is important for determining the fitness, survival, and growth of cyanobacteria in the laboratory as well as in large scale cultivation

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    circFL-seq, A Full-length circRNA Sequencing Method
    Authors:  Zelin Liu and Ence Yang, date: 04/20/2022, view: 1229, Q&A: 1
    [Abstract]

    Due to overlapping sequences with linear cognates, identifying internal sequences of circular RNA (circRNA) remains a challenge. Recently, we have developed a full-length circRNA sequencing method (circFL-seq) and computational pipeline, to profile ordinary and fusion circRNA at the isoform level. Compared to short-read RNA-seq, rolling circular

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    Identification of SARS-CoV-2 Neutralizing Antibody with Pseudotyped Virus-based Test on HEK-293T hACE2 Cells
    [Abstract]

    Neutralizing antibodies (NAbs) are of particular importance because they can prevent binding of the receptor binding domain (RBD) of the spike protein (S protein) to the angiotensin-converting enzyme 2 (ACE2) receptor present at the surface of human cells, preventing virus entry into the host cells. The gold standard method for detection of

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    CD8 T Cell Virus Inhibition Assay Protocol
    [Abstract]

    The human immunodeficiency virus (HIV)-1 viral inhibition assay (VIA) measures CD8+ T cell-mediated inhibition of HIV replication in CD4+ T cells and is increasingly used for clinical testing of HIV vaccines and immunotherapies. Different VIAs that differ in length of CD8:CD4 T cell culture periods (6–13 days), purity of CD4 cultures

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    Small Molecule Screening of Primary Human Acute Myeloid Leukemia Using Co-culture and Multiplexed FACS Analysis
    Authors:  Aurelie Baudet, Simon Hultmark, Fredrik Ek and Mattias Magnusson, date: 03/20/2022, view: 675, Q&A: 0
    [Abstract]

    Ex vivo culture of primary acute myeloid leukemia (AML) cells is notoriously difficult due to spontaneous differentiation and cell death, which hinders mechanistic and translational studies. To overcome this bottleneck, we have implemented a co-culture system, where the OP9-M2 stromal cells support the growth, but most notably limit the

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    An in vitro Blood-brain Barrier Model to Study the Penetration of Nanoparticles
    Authors:  Sezin Aday, Wen Li, Jeffrey M. Karp and Nitin Joshi, date: 02/20/2022, view: 4419, Q&A: 0
    [Abstract]

    The blood-brain barrier (BBB), a crucial protection mechanism in the central nervous system (CNS), is a selective barrier comprised of endothelial cells. It hampers the development of therapeutic and diagnostic tools for neurological diseases due to the poor penetration of most of these agents. Rationally engineered nanoparticles (NP) can

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    A Cell-based Screening Method Using an Intracellular Antibody for Discovering Small Molecules Targeting Hard-to-drug Proteins
    Authors:  Nicolas Bery and Terry H. Rabbitts, date: 02/20/2022, view: 2079, Q&A: 0
    [Abstract] Targeting hard-to-drug proteins, such as proteins functioning by protein-protein interactions (PPIs) with small molecules, is difficult because of the lack of well-defined pockets. Fragment or computational-based methods are usually employed for the discovery of such compounds, but no generic method is available to quickly identify small molecules ...
    NBD-lipid Uptake Assay for Mammalian Cell Lines
    Authors:  Sara Abad Herrera, Marta Grifell-Junyent and Thomas Günther Pomorski, date: 02/20/2022, view: 1873, Q&A: 0
    [Abstract]

    All eukaryotic cells are equipped with transmembrane lipid transporters, which are key players in membrane lipid asymmetry, vesicular trafficking, and membrane fusion. The link between mutations in these transporters and disease in humans highlights their essential role in cell homeostasis. Yet, many key features of their activities, their

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    Preparation and Cultivation of Colonic and Small Intestinal Murine Organoids Including Analysis of Gene Expression and Organoid Viability
    Authors:  Luisa Klemke, Joshua P. Blume, Tiago De Oliveira and Ramona Schulz-Heddergott, date: 01/20/2022, view: 2399, Q&A: 0
    [Abstract]

    Organoids are complex three-dimensional structures, which contain different cell types and help to overcome many limitations of conventional 2D cell culture techniques. Here, we present a protocol for the cultivation of murine matched-pairs of small intestinal and colonic epithelial organoids, and colonic tumor organoids derived from the chemical

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