Detection of HBD1 peptide in peripheral blood mononuclear cell subpopulations by intracellular flow cytometry

Abstract

Production of human β-defensin1 (HBD1) in response to LPS in monocytes, myeloid dendritic cells and plasmacytoid dendritic cells (PDC) was examined. Since PDC make up only 0.1–0.5% of the peripheral blood mononuclear cell population, we developed a method to determine HBD1 peptide levels using four-color flow cytometry, which can examine several cell surface or intracellular markers at once. Coupled with intracellular flow cytometry, we determined that PDC and monocytes only made significant amounts of HBD1 when exposed to >50 ng/ml LPS for 2 h. This response was limited to monocytes when ultrapure LPS was used, and was inhibited in PDC by chloroquine treatment.