Flow cytometry analysis

hADSCs were seeded at 25,000 cells/cm² density on TCP, on the Integra™ scaffold and on both sides of the MatriStem™ and the XenoMEM™ as described above and cultured for 14 and 21 days in α-MEM with 10% FBS and 1% PS at 5% CO₂ and 37 °C, replacing the media every 3 days. At each time point, cells were detached with 0.25% trypsin/ethylenediaminetetraacetic acid (EDTA) solution, filtered through a 40-μm cell strainer (ThermoFisher, Ireland), centrifuged at 300×g for 5 min, resuspended at 10⁶ cells/ml with 2% FBS and 0.05% sodium azide in PBS and kept on ice. One hundred microliters of each cell suspension (~ 10⁵ cells) were incubated with fluorescence labelled antibodies for the mesenchymal stem cells markers CD90⁺, CD73⁺, CD44⁺ (respective product codes 51-9007657, 51-9007649, 51-9007656, BD Bioscience, Ireland) and CD45⁻ (product code 46-0459-41, ThermoFisher, Ireland) and their correspondent isotype controls (CD90⁺, CD73⁺, CD44⁺ isotype cocktails, product codes 51-9007664, 51-9007655 , BD Bioscience, Ireland; CD45⁻ isotype, product code 46-4714-80, ThermoFisher, Ireland) for 30 min at 4 °C in dark. The cell suspensions were then centrifuged at 300×g for 5 min, washed in 2 ml of 2% FBS and 0.05% sodium azide solution and centrifuged as above. The supernatants were discarded by decantation and the cell pellets were resuspended in the remaining FBS and sodium azide solution with a vortex, and 5 μl of Sytox™ Blue (ThermoFisher, Ireland) were added to stain dead cells. Cell suspensions were then analysed using a BD FACS Canto™ II flow cytometer (BD Biosciences, Ireland). Analysis was carried out until 10,000 counts were reached and data were processed with the software FlowJo™ v10 (FlowJo™ LLC, USA).