Labeling hBM-MSCs with Molday ION and PKH26

The labeling of cells with Molday ION consisted of SPIO nanoparticles, and rhodamine purchased from BioPAL (Worcester, MA, USA) was performed as previously described by us.35 (link) Briefly, 100 μL of Molday ION was added to the 5×10⁵ hBM-MSCs cultured in 10 mL of MSCGM and incubated over 16 hours at 37°C in a humidified atmosphere containing 5% CO₂. After that, medium with label was removed, cells were washed with phosphate-buffered saline (PBS), fresh medium was added, and cells were cultured for 48 or 72 hours. The labeling of cells with PKH26 (Red Fluorescent Cell Linker Kits MINI26; Sigma-Aldrich Co., St Louis, MO, USA) was performed at room temperature (RT) for 5 minutes in the dark and blocked with fetal bovine serum (FBS), according to manufacturer’s instructions. The unincorporated stains were removed by hBM-MSCs centrifugation at 400× g for 10 minutes at 20°C–25°C using Eppendorf Centrifuge 5804R. hBM-MSCs were washed with Dulbecco’s PBS (DPBS) without Ca++ and Mg++ (Lonza) and subjected to additional centrifugation. The pellet was re-suspended, and cells were plated in 75 cm² polystyrene tissue culture flasks as described earlier.