A simple, robust flow cytometry-based whole blood assay for investigating sex differential interferon alpha production by plasmacytoid dendritic cells

Central to sex differences observed in outcome from infection and vaccination is the innate immune response, and specifically production of type I interferons by plasmacytoid dendtiric cells (pDCs), the main producers of IFN-α. Evaluation of IFN-α production by pDCs is therefore critical for studies of innate immune function. However, reliable measurement of pDC IFN-α is hampered by reduced cell yields and cytokine production after cryopreservation or after even short delays in stimulating freshly isolated cells. We here describe a simple yet robust method for measuring IFN-α production in pDCs that preserves cell activation and cytokine production through immediate stimulation of whole blood and subsequent maintenance at 37 °C. •Immediate whole blood stimulation detects differential IFN-α production by pDCs•Maintaining whole blood samples at 37 °C preserves maximal cytokine production•6 h incubation best captures differential pDC activation and cytokine production•Assay robustness is confirmed by low intra-donor temporal variability•Cell activation and cytokine production are substantially reduced by cryopreservation