Flow Cytometry Immunophenotyping Protocol

Surface and intracellular staining for flow cytometry analysis was performed as described previously (46 (link), 47 (link)). For surface staining, cells were incubated with relevant fluorochrome-labeled antibodies for 30 min at 4°C in the dark. For intracellular cytokine staining, cells were fixed and permeabilized using the Intracellular Fixation & Permeabilization buffer set (Invitrogen, USA) and stained with allophycocyanin (APC)-anti-IFN-γ, peridinin chlorophyll protein (PerCP)-Cy5.5-anti-IL-2, or fluorescein isothiocyanate (FITC)-anti-TNF-α (BD Biosciences, USA). Freshly isolated cells were used for all assays. Approximately 100,000 PBMCs were acquired for each sample using a BD FACS Canto II flow cytometer. Data analysis was performed using FlowJo software V10.0.7 (Tree Star, Ashland, OR, USA). Cell debris and dead cells were excluded from the analysis based on scatter signals and Fixable Viability Dye eFluor 506.