Facsymphony spectral analyzer

Fresh or frozen PBMCs isolated from the indicated patient groups were stimulated with antigen mixtures as above for 20–22h at 37°C, 5% CO_2. For intracellular staining and cytokine detection, the Brefeldin-A Golgi plug (Biolegend) was added at a 1:1000 concentration 2 hours after antigenic stimulation commenced. Cells were washed with PBS-1% BSA after incubation and incubated with the indicated antibodies for surface phenotyping by AIM assay or for intracellular cytokine staining (ICS; antibodies used described in Supplemental Table 1). Cells from each subject were left unstimulated in medium containing 5ng/mL IL-15 (“background”) or stimulated in the presence of the indicated antigens. Fixation and permeabilization was performed using Cytofix/Cytoperm (BD Biosciences). Surface staining was done in the dark at 4°C for 30 minutes, while ICS was done in the dark at RT for 45 minutes. Flow cytometry was conducted on 2–5×10⁵ cells per condition. Data was acquired on a BD FACSymphony Spectral analyzer and analyzed using FlowJo v10 (BD Biosciences) and SPICE-Pestle(71 (link)).