Selected early and senescent passage cells were defrosted from frozen vials in a water bath at 37 °C. The cells were counted to be of at least 105 cells per tube. The cell pellet was re-suspended in 200 μl of blocking buffer (0.5% BSA—bovine serum albumin, 2% FBS in 1× PBS) and incubated for 15 min at room temperature. FACS buffer (0.5% BSA, 0.05% Sodium Azide in 1× PBS) of 200 μl were added to the suspension and the solution was split into tubes with 50 μl each. Antibodies against MSC positive markers (Miltenyi Biotec): CD73-PE (Clone AD2), CD90-PerCP-Vio700 (Clone REA897), CD105-FITC (Clone 43A4E1) and negative markers (Viogreen): CD14 (Clone REA599), CD19 (Clone LT19), CD34 (Clone AC136), CD45 (Clone REA747), HLA-DR (Clone REA805) were added and solutions were incubated for 15 min at 4 °C in the dark. FACS buffer of 500 μl were added to each tube to wash off non-binding antibodies. Tubes of stained and unstained cells were spun down at 400 rcf for 5 min, re-suspended in 500 μl FACS buffer and the data were acquired by Attune Acoustic Focusing Flow Cytometer (Applied Biosystems). The FlowJo software (version 10.7) (http://www.flowjo.com/solutions/flowjo/downloads) was used for data analysis with debris excluded by gates, and the percentage of cell expressing these surface markers were also recorded.

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