Cd44 apc

Apoptosis assay was performed using Annexin V-FITC and propidium iodide staining kit (Keygen, Nanjing, China) according to the manufacturer’s protocol. To detect CD133⁺/CD44⁺ cells, CD133-PE (#130–090-853) and CD44-APC (#130–098-110) antibodies (Miltenyi Biotec.) were utilized to label cells. Then, labelled cells were subjected to flow cytometric analyses.

Flow cytometry analysis for hematopoietic and non-hematopoietic cell surface markers was performed on freshly isolated mononuclear cells and on adherent cell populations at p0 and p1. Cells were incubated with single or combinations of the following mouse monoclonal anti-human antibodies in PBS containing 1% fetal calf serum for 30 min at 4 °C: CD45-Alexa 488 (1:20, Beckman Coulter, Nyon, Switzerland), CD45-APC (1:5), CD73-PE-Cy7 (1:40), CD105-PE (1:40), CD90-BV421 (1:13), CD146-PeCy7 (1:20), HLA-DR-PE (1:5), CD19-PE (1:5), CD11b-PE (1:5, all BD Bioscience), CD44-APC (1:10), CD34-PE (1:10, both Miltenyi Biotec, Bergisch-Gladbach, Germany), NG2-PE (1:10), PDGF-rβ-Alexa 700 (1:20, both R&D Systems). Unstained isotype (IgG1-PeCy7 (1:20, BD Bioscience)) and single antibody controls were included. Flow cytometry was performed using a BD Aria III, and a minimum of 25,000 events were acquired for each sample, and data were analyzed using BD FACS Diva 6.1.3. Appropriate compensation settings and gating were applied in order to account for cellular debris, cell doublets, spectral overlap, and autofluorescence.

Expression of cell surface markers CD90, CD44, and CD49f was determined using a MACSQuant flow cytometer using the MACSQuant Analyzer 10 software (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and labelled antibodies CD90-PE, CD44-APC, and CD49f-FITC (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany).