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

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...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2020-05, Vol.92 (9), p.6200-6206
Main Authors: AbuElela, Ayman F, Al-Amoodi, Asma S, Ali, Amal J, Merzaban, Jasmeen S
Format: Article
Language:English
Subjects:
Analytical chemistry
Animals
Antibodies - chemistry
Antibodies - immunology
Assaying
Cell adhesion
Cell adhesion & migration
Cell Line
Cell migration
Chemistry
CHO Cells
Confocal microscopy
Cricetinae
Cricetulus
Deglycosylation
E-selectin
E-Selectin - immunology
E-Selectin - metabolism
Endothelial cells
Flow chambers
Fluorescence
Fluorescent Dyes - chemistry
Glycan
Homing
Homing behavior
Humans
Image acquisition
Immunoassay
Leukemia
Ligands
Line spectra
Microscopy, Confocal - methods
Multiplexing
Parallel plate flow cells
Polysaccharides
Real time
Selectins
Shear stress
Stem Cells - cytology
Stem Cells - metabolism
Tethering
Time-Lapse Imaging
Wavelengths
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Summary: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.
ISSN:0003-2700
1520-6882
DOI:10.1021/acs.analchem.9b04549