Quantitative Analysis of Siglec Ligands by Flow Cytometry

Abstract

AbstractSiglecs (sialic acid‐binding, immunoglobulin superfamily, lectins) are a family of transmembrane receptor‐type glycan recognition proteins in vertebrates that are primarily expressed on leukocytes and regulate immune responses. Siglecs are involved in several diseases, such as cancer and neurodegenerative diseases. Most Siglecs suppress the activation of leukocytes by recognizing ligands containing sialic acid, a group of acidic sugars commonly found in vertebrate glycans, but rare among microbes. Siglec ligands are critical in the interaction between leukocytes and target cells. The abundance of the Siglec ligand is influenced by both the abundance of the glycoconjugate carrier (glycoprotein or glycolipid) and that of the terminal glycan epitope directly recognized by the Siglec. Therefore, a direct approach to evaluate the expression level of a Siglec ligand on cells of interest is to analyze the binding of recombinant Siglec protein to these cells. In this article, we describe a protocol for semi‐quantitatively analyzing the expression level of Siglec ligands via flow cytometry using recombinant Siglec‐Fc fusion protein. Support protocols describe how to remove sialic acids from the cell surface with sialidase under mild conditions to demonstrate the sialic acid dependence of Siglec binding, and the preparation of recombinant Siglec‐Fc fusion proteins by transient transfection of mammalian cells. © 2023 Wiley Periodicals LLC.Basic Protocol: Quantitative analysis of Siglec ligands on mammalian cells via flow cytometry with recombinant Siglec‐Fc fusion proteinSupport Protocol 1: Sialidase treatment of mammalian cellsSupport Protocol 2: Preparation of recombinant Siglec‐Fc fusion protein via transient transfection of mammalian cells