In vitro stimulation and detection of IFNα production in human plasmacytoid dendritic cells

Type-1 interferons (IFNs), including IFNα/β, are a family of cytokines produced rapidly upon pathogen encounter and crucial for bridging innate and adaptive immunity. IFNα has been widely appreciated as a multifunctional cytokine involved particularly in early immune responses against viral, bacterial, and parasitic infections. Although most cells may be competent to produce IFNα during specific conditions, plasmacytoid dendritic cells (PDCs) are unique in their capacity to produce rapid and robust levels in response to various pathogens. PDCs to a great extent utilize toll-like receptor (TLR) 7 and 9, localized in early endosomes, to sense pathogen-associated nucleic acids, and initiate the signaling cascade leading to induction of IFNα. Here, we provide basic protocols for the detection of IFNα in individual immune cells, particularly PDCs, using flow cytometry. We discuss the key elements for successful isolation of PDCs, stimulation, immunostaining, and identification of IFNα producing cells.