Characterization of Stromal Cell Purity

Purity, identity and viability of stromal cells were characterized by flow cytometry as previously described^{39 (link)}. In brief, 5 × 10⁵ cells at passage 1 were resuspended in 50 µL phosphate buffered saline (PBS, Sigma Aldrich). Cells were mixed with 0.5 µL viability dye (Fixable Viability Dye eFluor™ 520, eBioscience, Thermo Fisher Scientifics, Waltham, MA, USA) and antibody mastermix for CD73, CD19, CD14, CD34, CD45, HLA-DR (all Becton Dickinson BD Biosciences, Franklin Lakes, NJ, USA), CD90 (Beckman Coulter, Brea, CA, USA) and CD105 (Life Technologies Corporation, Frederick, MD, USA). For determination of F-heparin internalization, 1 × 10³ cells per cm² were seeded. After 48 hours, cells were incubated for three hours with medium supplemented with 10% pHPL either without heparin, with 2 IU/ml unlabeled heparin (Biochrom) or with 2 IU/ml fluoresceinamine-labeled sodium heparin (F-heparin, PG Research, Tokyo, Japan). After washing cells twice with PBS, 2 × 10⁵ cells were resuspended in 100 µL 7AAD viability dye mastermix (BD). All cells were measured immediately (BD LSRFortessa™A) and results were analyzed with Kaluza Analysis Software (Beckman Coulter).