Ssea4 pe

The cells were detached, dissociated into single cells using dispase (Sigma-Aldrich), and resuspended in fluorescence-activated cell sorting (FACS) buffer (phosphate-buffered saline PBS, 1% BSA; 5 × 10⁵ cells/100 μl FACS buffer). For cell-surface staining the cells were stained with primary antibodies CD73-PE (130-095-182, Miltenyi), CD105-FITC (130-094-926, Miltenyi), CD90- PE-VIO770 (130-099-295, Miltenyi), CD45-PE (130-098-141, Miltenyi), CD34-FITC (130-098-139, Miltenyi), CD24-FITC (130-098-861, Miltenyi), SSEA4-PE (FAB1435P, R&D), and TRA1-60-PE (09-0009, Stemgent) for 1 hour at 4 °C. For intracellular FACS staining, i.e. OCT4-A (130-105-606, Miltenyi) and OCT4-B (IC1759P, R&D), the cells were washed once in FACS buffer, fixed for 10 min in 0.01% paraformaldehyde (PFA), washed twice with PBS, resuspended in permeabilization buffer (PBS, 1% Triton) and stained as describe above. Cells were then washed twice with PBS, resuspended in FACS buffer and analysed by FACScalibur flow cytometry (CyAn ADP, Beckman Coulter). Collected data were analyzed with the Flowjo v.10 software package (Treestar, Ashland, OR, USA). Negative control was immunoglobulin G (IgG) primary antibody-specific isotypes.

Cultures of hASCs-M, hASCs-T, hASCs-TS and hASCs-TE were phenotypically characterized by flow cytometry (FC). After trypsinization, cells were resuspended with FC buffer (pH 7.2 PBS, BSA 0.5%, sodium azide 0.02%) at a concentration of 0.1 x 10⁶/100 μl. Cells were incubated with fluorescently labeled antibodies for 30 minutes at room temperature in a dark room and then washed with FC buffer to remove non-conjugated antibodies. Fluorescein isothiocyanate (FITC-F) or phycoerythrin (PE) conjugate-antibodies were used: CD90PE, CD105PE, CD34PE, CD45F, CD44F, CD106PE, CD146PE (Immunotech®, Milan, Italy), HLA-IF, HLA-IIPE (Biolegend®, Italy), CD73PE, SSEA-4PE (R&D®, Milan, Italy). Epics "XL-MCL" (Beckman Coulter, USA) flow cytometer was used for analyzing fluorescent immunophenotypic marker signals. At least 10,000 events for test samples were acquired. Sample histogram elaboration was performed with EXPO 32 software to assess fluorescent distribution.