Fluorescent Multiplex Cell Rolling Assay: Simultaneous Capturing up to Seven Samples in Real-Time Using Spectral Confocal Microscopy.
Type
ArticleKAUST Department
Cell Migration and Signaling LaboratoryBioscience Program
Biological and Environmental Sciences and Engineering (BESE) Division
Date
2020-04-08Online Publication Date
2020-04-08Print Publication Date
2020-05-05Embargo End Date
2021-04-21Submitted Date
2019-10-06Permanent link to this record
http://hdl.handle.net/10754/662667
Metadata
Show full item recordAbstract
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.9b04549Sponsors
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 chemistryPubMed ID
32264668Additional Links
https://pubs.acs.org/doi/abs/10.1021/acs.analchem.9b04549ae974a485f413a2113503eed53cd6c53
10.1021/acs.analchem.9b04549
Scopus Count
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