Luminex 200 platform

According to the manufacturer’s instructions of the Human Cytokine/Chemokine Panel MILLIPLEX® MAP kits (Cat. No. HCYTOMAG-60K: IFN-γ, IFN-α2, IL-1β, IL-1α, IL-8, IL-4, IL-6, IL-10, IL-12(p70), IL-15, IL-17A, TNF-α) (Merck Millipore, Germany), serum concentrations of multiple cytokines were measured by a Luminex200 platform (Merck Millipore, Germany). In brief, the plate was pretreated with 200 μl wash buffer for10 min. Next, 25 μl serum of each sample, 25 μl assay buffer and 25 μl mixed beads were added to sample wells. The background wells, QC wells and standard wells were added with corresponding reagents following the instructions. The plate was sealed and incubated with shaking at 4 °C overnight. The next day, the plate was washed twice with wash buffer and 25 μl detection antibodies were added for a 1 h incubation at room temperature. After washed twice with wash buffer, each well was incubated with 25 μl Streptavidin Phycoerythrin for 30 min. After two wash steps, 150 μl sheath fluid was added and the data were collected by the Luminex200 platform. All samples were measured in duplicate.

T3 concentration in NMed suspension was also confirmed in buffer at pH 7.4 and pH 6.8, using an xMap commercial kit (Merck-Millipore, Milan, Italy, cat: RTHYMAG-30K) and the Luminex 200 platform. Briefly, samples were incubated overnight with micromagnetic beads conjugated with a specific primary antibody. After washing three times, a secondary antibody and Streptavidin-Phycoerythrin complex were added to the wells. The median fluorescent intensity (MFI) data obtained by Luminex^®200^TM were used to calculate analyte concentrations in samples using a 5-parameter logistic curve-fitting method. Results were expressed in ng/mL.

Cytokines in mice sera, DCs, or mouse DC supernatants were quantified using multiparametric Luminex kits. In brief, interferon gamma (IFN-γ), IL-2, IL-4, IL-6, IL-10, IL-12 (p70), IL-17A, KC/CXCL1, MIP-2, and TNF-α levels in mice serum samples were quantified using the Luminex 200 platform with a magnetic system (Milliplex MAP Mouse High Sensitivity T Cell Magnetic Bead Panel, EMD Millipore Corporation, Billerica, MA, USA) following the manufacturer's instructions. Cytokine concentrations are expressed as the average of three replicates in pg/mL ± SD. Similarly, cytokines in the human sera of COVID-19 patients, and vaccinated or control volunteers were quantified using the multiparametric Luminex kit (Milliplex human 1xl HSTCMAG-28SK including the following cytokines: IFN-γ, IL-10, IL-17A, IL-2, IL-4, IL-6, IL-8, TNF-α, EMD Millipore Corporation) following the manufacturer´s instructions.