Dioc18 7 dir

For cell labeling, unprimed and primed CBti MSCs in selected experiments were labeled using lipophilic carbocyanine DiOC18(7) (DiR) (ThermoFisher), following the Manufacturer's recommendations. For in vivo and ex vivo tissue detection confirmation, three animals each were dosed via tail vein injections with 2 × 10⁶ of labeled primed or unprimed CBti MSCs. At 24, 48, and 72 h following the injection animals were subjected to imaging (IVIS Lumina XR Imagine System). After each time point, mice were sacrificed, and selected tissues were harvested. Tissues were analyzed for the presence of cells by fluorescence to confirm that DiR signals were representative of the cell location. Signal quantitation in photons/second was performed by determining the photon flux rate within standardized regions of interest using Living Image software (Caliper).

Acceptor cells were cultured following standard culturing conditions. Cytoplasm was stained with ViaFluor®488 proliferation dye (Biotium, Fremont, CA, USA) 24h prior to the uptake experiment and seeded at 3 x 103 cells per well in a 384-well plate (PerkinElmer, Waltham, MA, United States). The following day, cells were stained with NucBlue™ Live ReadyProbes™ Reagent (Hoechst 33342) (Thermo Fisher Scientific), washed and maintained in regular growth medium. Langendorff perfusate-derived EVs were stained with DiOC18(7) (DiR) (Thermo Fisher Scientific). Stained EVs were added to cell medium at a final concentration of 13 ng protein/μl. Cells were imaged for 16 h using an Opera Phenix™ system (PerkinElmer). Analysis of internalization of EVs was performed using Harmony v.4.9 (Perkin Elmer).