Anti cd3 viogreen

Following 48 h, whole blood incubation, RBC lysis buffer (Thermo Fisher Scientific) was used to lyse the red blood cells. After washing with PBS, cells were stained with anti-CD3-Viogreen, anti-CD69-APCVio770, anti-CD95-PEVio770, anti-CD25-PE, anti-CD71-FITC, and anti-CD154-VioBlue (Miltenyi Biotec). The samples were measured after a final washing step, using a MaqsQuant10 analyzer. Before measurement, propidium iodide (PI) (Miltenyi Biotec) was added to assess viability. Analysis of the cell populations was performed with Flowlogic software (Inivai Technologies, Mentone VIC, Australia). For each time point the unstimulated samples were used to set the correct gating. The gating strategy is shown in Figure A1 in Appendix A.

NK cells were isolated via negative magnetic bead selection using NK cell isolation kit (Miltenyi, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. A fraction of NK cells were washed twice in saline solution and stained with anti-CD56 BV421 (Clone NACM16.2), anti-CD3 VioGreen (Miltenyi, Bergisch Gladbach, Germany; Clone REA613), anti-CD16 FITC (Miltenyi, Bergisch Gladbach, Germany; Clone REA423), anti-CD32 PE (Miltenyi, Bergisch Gladbach, Germany; Clone REA997), and anti-CD64 APC (Miltenyi, Bergisch Gladbach, Germany; Clone REA978). Samples were stained in the dark at 4°C and were acquired using a FACSCelesta flow cytometer (BD Biosciences, San Diego, USA). Post-acquisition editing and data analysis was conducted using FlowJo^® 10.6.2 software (FlowJo LLC, Ashland, USA). NK cells were defined as CD3^- CD56⁺ and/or CD16⁺ lymphocytes.

PBMCs were stimulated with 2.5 µg/mL Concanavalin A in the absence or presence of MSCs for 5 days. Then, PBMCs were collected and stained with the following antibodies: Anti-CD3-VioGreen, anti-CD19-PEVio770, anti-CD8-APC (Miltenyi Biotec, Bergisch Gladbach, Germany), or corresponding isotype controls following the manufacturer’s instructions. Data were acquired using the MACSQuant^® analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany). Data were analyzed using FlowJo^TM v10.6.2 Software (BD Life Sciences, Ashland, OR, USA).