4.9. Cytokine Measurement
Serum cytokine levels were determined using a flow cytometry bead-based LEGENDplex™ HU Essential Immune Response Panel (BioLegend, San Diego, CA, USA), a 13-plex human panel to analyze the following cytokines and chemokines: IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-17A CCL2 (MCP-1) CXCL10 (IP-10), IFN-γ, TGF-β1, and TNF-α. The assay was performed following the manufacturer’s protocol; briefly, samples were diluted 2-fold with assay buffer and 50 mL of sample or standard was mixed with 25 μL of previously vortexed mixed beads in a V-bottom plate, then incubated for 2 h, centrifuged at 250 rpm for 5 min, and washed two times with 200 μL wash buffer, 25 μL of detection antibody were added and incubated as above, and without washing, 25 μL of SA-PE were added and incubated for 30 min, and afterward, centrifugation and washing steps were repeated, adding a final 150 μL of wash buffer to each well before being individually transferred to microtubes for the acquisition in a BD FACS AriaTM Fusion (BD Biosciences, San Jose, CA, USA). Analytes concentrations were calculated using the LEGENDplex Data Analysis SoftwareCatalog 740932 Version 9. Software GraphPad Prism version 9.4.0.