Cd90 pe vio770

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.