Cytometry analysis software

FACS analysis was used to measure cell cycle progression. After treatment with JQ1 for 24 h, cells were harvested, spun down at 4°C and washed before being fixed with 70% ethanol. RNA was degraded with RNAase and DNA stained with propidium iodide. Samples were analysed on a BD FACS Canto II (BD Biosciences, Oxford, UK). Histograms were generated and cell cycle analysis was performed using FlowJo cytometry analysis software (FlowJo, LLC, Ashland, OR, USA).

To assess the levels of chemokines – IL-8/CXCL-8, MCP-1/CCL-2, MIG/CXCL-9, IP-10/CXCL-10 and cytokines – TNF, INF-γ, IL-2, IL-4, IL-5 and IL-10 – in serum samples from 17DD-YF vaccinees, Cytometric Bead Array kits (BD Biosciences, California, USA) were used according to manufacturer’s protocol and adapted as described previously [14 (link)]. Analysis of raw data was performed using the FlowJo cytometry analysis software (FlowJo, Stanford, USA) and the mean fluorescence intensity (MFI) of each bead cluster was evaluated to calculate the each cytokine concentration in the sera of patients. Cytokines concentrations were extrapolated according to the standard curve created by serial dilutions of the positive control. The final data for the kinetics timeline design were expressed as fold changes based on the baseline concentration for all chemokines and cytokines tested.