Affiliation:
1. Department of Biomedical Engineering New Jersey Institute of Technology Newark New Jersey
2. Department of Chemical and Materials Engineering New Jersey Institute of Technology Newark New Jersey
Abstract
AbstractThe leukocyte adhesion cascade governs the recruitment of circulating immune cells from the vasculature to distal sites. The initial adhesive interactions between cell surface ligands displaying sialyl‐LewisX (sLeX) and endothelial E‐ and P‐selectins serve to slow the cells down enough to interact more closely with the surface, polarize, and exit into the tissues. Therefore, precise microfluidic assays are critical in modeling how well immune cells can interact and “roll” on selectins to slow down enough to complete further steps of the cascade. Here, we present a systematic protocol for selectin mediated rolling on recombinant surfaces and endothelial cell monolayers on polyacrylamide gels of varying stiffness. We also describe step‐by‐step the protocol for setting up and performing the experiment and how to analyze and present the data collected. This protocol serves to simplify and detail the procedure needed to investigate the initial selectin‐mediated interactions of immune cells with the vasculature. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Preparing dishes for cell rolling experimentsBasic Protocol 2: Fabrication of polyacrylamide gels for cell rolling experimentsAlternate Protocol 1: Protein conjugation with N6 linkerAlternate Protocol 2: HUVEC culturing for monolayersBasic Protocol 3: Conducting cell rolling experiments on polyacrylamide gelsBasic Protocol 4: ImageJ analysis of cell rolling moviesBasic Protocol 5: Quantification of Fc site density on a surface (e.g., for Fc chimeras)