Analyzing T-Cell Plasma Membrane Lipids by Flow Cytometry

Plasma membrane lipid rafts are highly ordered membrane microdomains enriched for glycosphingolipids and cholesterol, which play an important role during T-cell antigen receptor (TCR) signaling. Our previous work has demonstrated that plasma membrane lipid composition is an important determinant of human CD4+ T-cell function and that defects in lipid raft expression contribute to CD4+ dysfunction in patients with autoimmunity. In this chapter we share three flow cytometry-based methods to quantitatively analyze plasma membrane lipid composition in primary human CD4+ T cells. We describe the quantification of glycosphingolipid expression using cholera toxin subunit B, cholesterol expression using filipin staining, and membrane "lipid order" using di-4-ANEPPDHQ. These methods can easily be adapted to analyze different cell types.