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.
Highlights
The parallel plate flow chamber assay is widely utilized to study physiological cell−cell adhesive interactions under dynamic flow that mimics the bloodstream
The analysis of rolling capabilities of cells has been largely studied in vitro using parallel plate flow chamber (PPFC)-based assays.[14−16,24−34] These assays mimic cells migrating through the circulation where flowing cells are infused under shear stress over a monolayer of a substrateexpressing cells (or a polyvinylidene difluoride (PVDF)
Membrane boasting immunoprecipitated ligands of interest adsorbed on its surface) seeded on the surface of one of the parallel plates of the setup.[14,15,24,28−30,32,33] Using a bright-field microscope and video recording system to image the process, each sample or cell type is analyzed in a singleplex mode as it is perfused over a monolayer of substrate-expressing cells under defined shear stresses controlled by a syringe pump
Summary
The parallel plate flow chamber assay is widely utilized to study physiological cell−cell adhesive interactions under dynamic flow that mimics the bloodstream. The materials required for this assay included (Figure S1): (i) a cell suspension of a mixture of cells (e.g., KG-1a cells) tagged with UFIs (Figures 1A and S2; see Fluorescent Multiplex Cell Rolling (FMCR) in the Experimental Section); (ii) a monolayer of substrateexpressing cells (e.g., E-selectin-expressing CHO cells; see “Preparation of substrate expressing cells for FMCR” in Methods of the Supporting Information); (iii) a parallel plate flow chamber with the lower plate (a coverslip) coated with a monolayer of the substrate-expressing cells and placed on the stage of an inverted microscope; (iv) an inverted microscope equipped with laser-scanning spectral confocal system; (v) a computer for time-lapse image capturing and storage; and (vi) a syringe pump for cell infusion under a defined stepwise program of shear stress magnitudes.
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