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    Fluorescent Multiplex Cell Rolling Assay: Simultaneous Capturing up to Seven Samples in Real-Time Using Spectral Confocal Microscopy.

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    Type
    Article
    Authors
    AbuElela, Ayman F
    Al-Amoodi, Asma S
    Ali, Amal J. cc
    Merzaban, Jasmeen cc
    KAUST Department
    Cell Migration and Signaling Laboratory
    Bioscience Program
    Biological and Environmental Sciences and Engineering (BESE) Division
    Date
    2020-04-08
    Online Publication Date
    2020-04-08
    Print Publication Date
    2020-05-05
    Embargo End Date
    2021-04-21
    Submitted Date
    2019-10-06
    Permanent link to this record
    http://hdl.handle.net/10754/662667
    
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    Abstract
    The parallel plate flow chamber assay is widely utilized to study physiological cell-cell adhesive interactions under dynamic flow that mimics the bloodstream. In this technique, the cells are perfused under defined shear stresses over a monolayer of endothelial cells (expressing homing molecules, e.g., selectins) or a surface (expressing recombinant homing molecules). However, with the need to study multiple samples and multiple parameters per sample, using a traditional bright-field microscope-based flow assay allows only one sample at a time to be analyzed, resulting in high interexperiment variability, the need for normalization, waste of materials, and significant consumption of time. We developed a multiplexing approach using a three-color fluorescence staining method, which allowed for up to seven different combination signatures to be run at one time. Using this fluorescent multiplex cell rolling (FMCR) assay, each sample is labeled with a different signature of emission wavelengths and mixed with other samples just minutes before the flow run. Subsequently, real-time images are acquired in a single pass using a line-scanning spectral confocal microscope. To illustrate the glycan-dependent binding of E-selectin, a central molecule in cell migration, to its glycosylated ligands expressed on myeloid-leukemic cells in flow, the FMCR assay was used to analyze E-selectin-ligand interactions following the addition (fucosyltransferase-treatment) or removal (deglycosylation) of key glycans on the flowing cells. The FMCR assay allowed us to analyze the cell-adhesion events from these different treatment conditions simultaneously in a competitive manner and to calculate differences in rolling frequency, velocity, and tethering capability of cells under study.
    Citation
    AbuElela, A. F., Al-Amoodi, A. S., Ali, A. J., & Merzaban, J. S. (2020). Fluorescent Multiplex Cell Rolling Assay: Simultaneous Capturing up to Seven Samples in Real-Time Using Spectral Confocal Microscopy. Analytical Chemistry. doi:10.1021/acs.analchem.9b04549
    Sponsors
    We would like to thank Ms. Samar A. Rustum and Ms. Umme Habiba for their support in the management of the lab along with the entire Cell Migration and Signaling Laboratory for support and discussions. This research was supported by a King Abdullah University of Science and Technology (KAUST) Faculty Baseline Research Funding Program to J.S.M.
    Publisher
    American Chemical Society (ACS)
    Journal
    Analytical chemistry
    DOI
    10.1021/acs.analchem.9b04549
    PubMed ID
    32264668
    Additional Links
    https://pubs.acs.org/doi/abs/10.1021/acs.analchem.9b04549
    ae974a485f413a2113503eed53cd6c53
    10.1021/acs.analchem.9b04549
    Scopus Count
    Collections
    Articles; Biological and Environmental Science and Engineering (BESE) Division; Bioscience Program

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