Lectin Flow Cytometry for Cell Surface Glycan Profiling: An Accessible Approach to Glycophenotyping

Lectins are widely used to detect glycan-associated features on the cell surface; however, their implementation in flow cytometry often requires empirical optimization and methodological considerations that are not always systematically described. Here, we provide a practical framework for implementing lectin-based glycophenotyping in flow cytometry, addressing both the experimental decisions involved in assay optimization and validation and its integration into conventional multiparametric workflows, highlighting its potential to address more complex biological questions. Using illustrative examples from murine splenocytes, we discuss key aspects of lectin titration, fluorochrome selection, negative controls, and specificity validation, and demonstrate the compatibility of lectin staining with commonly used flow cytometry applications, including viability assessment, immunophenotyping, intracellular cytokine detection, and proliferation analysis. By integrating methodological considerations with representative applications, this Technical Note provides a practical resource to guide experimental decision-making and facilitate the incorporation of lectin-based glycophenotyping into multiparametric flow cytometry workflows.