Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns

Abstract

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new devices for label-free separation and analysis of cells 1 . Such devices may use lateral displacement for continuous-flow separation, or receptor patterns that modulate adhesion to distinguish between different cell phenotypes or levels of receptor expression. Understanding the nature of cell rolling trajectories on receptor-patterned substrates is necessary for engineering of the substrates and design of such devices.Here, we demonstrate a protocol for studying cell rolling trajectories on asymmetric receptor patterns that support cell rolling adhesion 2 . Well-defined, μm-scale patterns of P-selectin receptors were fabricated using microcontact printing on gold-coated slides that were incorporated in a flow chamber. HL60 cells expressing the PSGL-1 ligand 3 were flowed across a field of patterned lines and visualized on an inverted bright field microscope. The cells rolled and tracked along the inclined edges of the patterns, resulting in lateral deflection 1 . Each cell typically rolled for a certain distance along the pattern edges (defined as the edge tracking length), detached from the edge, and reattached to a downstream pattern. Although this detachment makes it difficult to track the entire trajectory of a cell from entrance to exit in the flow chamber, particle-tracking software was used to analyze and yield the rolling trajectories of the cells during the time when they were moving on a single receptor-patterned line. The trajectories were then examined to obtain distributions of cell rolling velocities and the edge tracking lengths for each cell for different patterns.This protocol is useful for quantifying cell rolling trajectories on receptor patterns and relating these to engineering parameters such as pattern angle and shear stress. Such data will be useful for design of microfluidic devices for label-free cell separation and analysis. Using microcontact printing (μCP) 4-7 to make alternating self-assembled monolayers (SAMs) of PEG molecules on the gold-coated glass slides: Fabricate microcontact printing polydimethylsiloxane (PDMS) stamps that defined the receptor patterns with inclination angle of α = 10°by an SU-8 molding process. Clean the gold surface with piranha solution (3:1 mixture of sulfuric acid to 30% hydrogen peroxide) for 20 minutes and then rinse the surface with copious DI water at 24.5°C prior to use. Ink the PDMS stamp with 5mM PEG solution in ethanol. Dry the stamp in air for 20 minutes. Gently put the stamp on the gold surface for 40 sec and make sure there is a good contact between the gold surface and the stamp. No excess pressure is required. Rinse the surface with ethanol and dry it under a stream of N2. 2. Incubate the substrate within P-selectin solution (15 μg/mL in DPBS) using a perfusion chamber (Electron Microscopy Sciences) for 3 hours at 24.5°C to pattern the remaining areas with P-selectin. Rinse the surface w...

How Mouse Macrophages Sense What Is Going On

“…BAFF can be made by macrophages in soluble and membrane form, and membrane BAFF may (reverse) signal to promote inflammatory mediators ( 108 ), but the roles of the receptors are not well understood. Recent studies suggest that TACI or BCMA signals can also promote pro-inflammatory cytokines/mediators ( 109 ). TACI activity may also suppress differentiation of macrophages into the M2 phenotype, allowing enhanced clearance of intracellular organisms ( 110 ).…”

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

“…In the place of the conventional bright-field microscope used in the PPFC assay, − ,− the FMCR assay uses a confocal laser-scanning microscope (Figure S1) that allows the use of up to seven samples simultaneously through the combination of three fluorophores and the use of three detection filters. In addition, 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 A and S2; see Fluorescent Multiplex Cell Rolling (FMCR) in the Experimental Section); (ii) a monolayer of substrate-expressing 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.…”

“…To date, the analysis of rolling capabilities of cells has been largely studied in vitro using parallel plate flow chamber (PPFC)-based assays. − ,− These assays mimic cells migrating through the circulation where flowing cells are infused under shear stress over a monolayer of a substrate-expressing 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. ,,,− ,, 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 (or a substrate-coated surface) under defined shear stresses controlled by a syringe pump. Although a powerful tool, its design has a number of inherent technical limitations, including interexperimental variations stemming from pipetting errors and cell count errors that result in variations in the numbers of substrate-expressing cells in a given plate (seeding variation) and differences in the expression of substrates among cells (substrate expression disparity).…”

Microfluidic auto-alignment of protein patterns for dissecting multi-receptor crosstalk in platelets

“…1b). This new approach of micro-patterning allows the placement of target ligands at well-defined locations, which can be useful for studies of cell adhesion mediated by a range of receptor–ligand species 12,13 and for certain biological assays such as combinatorial screening 12 and cell sorting 13 . As a demonstration, we applied this method to interrogate the activation of platelets by GPIb—ligand binding, using α IIb β 3 activation and expression of P-selectin as readouts.…”