Facscanto 1

Cells were washed in PBS, prestained with fixable viability dye (eBioscience), fixed in 4% PBS‐buffered formalin, permeabilized using 0.2% Saponin and stained with antibodies in PBS. Flow cytometry was performed by FACSCanto I (Becton Dickinson) and data were analyzed using FlowJo (Tree Star).

2.5 × 10⁵ PBMCs were seeded in 96-well plates with RPMI 1640 (Gibco Cat # 11875119) supplemented with 10% FBS and 1% PS (Sigma Aldrich) and incubated with monensin (Golgistop; BD Biosciences Cat # 554715) for 5 h. After that, PBMCs were stained with anti-CD11b APC/Cy7 (BioLegend Cat # 10,225, RRID: AB_830641) anti-CD14-PerCP (BioLegend Cat # 325631, RRID: AB_2563327), anti-CD16-FITC (BioLegend Cat # 302005, RRID: AB_314205). Live cells were distinguished using Fixable Viability Dye eFluor™ 450 (eBioscience Cat # 65-0863-14). After staining of surface markers, cells were fixed and made permeable according to the manufacturer's instructions BD Cytofix/Cytoperm Kit (BD Bioscience Cat # 554715). Then, the cells were stained with anti-human IL-12-biotin (ThermoFisher Cat # AHC7129, RRID: AB_2536290) plus PE-conjugated Streptavidin (BioLegend Cat #405203), and acquired using a FACS Canto I (Becton Dickinson). All analysis was carried out with FlowJo software (Tree Star).

Wharton's Jelly human Mesenchymal Stem Cells (WJ-hMSCs) were isolated from umbilical cords from full-term deliveries, which were collected at the Gynaecology Unit of the Azienda Ospedaliera Universitaria-Pisa Hospital. Wharton's jelly (WJ) was separated from the cord vessels and placed in 6-well dishes containing alpha-minimum essential medium (α-MEM; GIBCO) supplemented with 20% fetal bovine serum (FBS; Euroclone, Italy) and incubated at 37°C with 5% humidified CO₂. Fresh medium was added twice a week up to 90% confluence. MSCs were then harvested with 0.25% trypsin and 1 mM EDTA solution (Cambrex, Italy) and re-plated at 8.000 cells/cm². Successive passages were performed in DMEM medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 10 U/ml penicillin and 10 mg/ml streptomycin. The expanded cells were characterized after the primoculture (P0) by flow cytometric analysis (FACS Canto I, Becton Dickinson CA, USA) of specific surface antigens (CD14−, CD34−, CD20−, CD45−, CD73+, CD90+ and CD105+) according to the mesenchymal immunophenotype. Cells were used within the 8th passage. In order to perform single-cell experiments, WJ-hMSCs were seeded at the final concentration of 30 × 10⁴ cells/cm² and kept in humidified atmosphere until experiment time.