Quantification of Mesenchymal Stem Cells

MSCs in primary tissue or culture were quantified as previously described [5 (link), 13 (link)]. Briefly, cells were labeled with a MSC Phenotyping Cocktail (anti-CD14 PerCP, anti-CD20-PerCP, anti-CD34-PerCP, anti-CD45-PerCP, anti-CD73-APC, anti-CD90-FITC, and anti-CD105-PE) or Isotype Control Cocktail (Mouse IgG1-FITC, Mouse IgG1-PE, Mouse IGG1-APC, Mouse IgG1-PerCP, and Mouse IgG2a-PerCP) included in the human MSC Phenotyping kit (Miltenyi) in a volume of 100 μl per 1×10⁶ cells for 10m at 4°C according to the manufacturer's instructions. Anti-HLA-DR-PerCP (Miltenyi) was also added to the MSC Phenotyping Cocktail. Analysis was performed using a BD FACSCalibur flow cytometer. All compensation controls were performed using anti-EpCAM antibodies directly conjugated to FITC, PE, APC, or Biotin followed by anti-Biotin-PerCP on aliquots of the same cell suspension to ensure proper gating and instrument settings.