Cytomics flow cytometer

For immunophenotyping, MSCs were stained with fluorescein isothiocyanate- (FITC-) or phycoerythrin- (PE-) conjugated monoclonal antibodies: CD14-FITC, CD29-FITC, CD31-PE, CD34-FITC, CD44-PE, CD45-PE, CD73-PE, CD90-FITC, CD105-PE, and CD106-FITC (all from BD Pharmingen, San Diego, CA, USA). Additionally, FITC- and PE-conjugated isotype controls were used as negative controls. Briefly, cultured MSCs were harvested and stained with the antibodies for 20 min at 4°C. Subsequently, the stained cells were washed with phosphate buffered saline (PBS) and fixed with 1% paraformaldehyde (Biosesang, Seongnam, Korea). The cells were analysed using a flow cytometer (Cytomics Flow Cytometer; Beckman Coulter, Fullerton, CA, USA).

The cultured MSCs at passage 7 were removed from uncoated and PLL-coated plates. Harvested cells washed with PBS and fixed with cold 70% ethanol while minimizing clumping. After 30 min at 4°C, the cells were washed with PBS and stained with propidium iodide. Propidium iodide fluorescence was then examined using the Cytomics Flow Cytometer (Beckman Coulter).

Human adipose tissue-derived MSCs were obtained from healthy donors with the approval from the research ethics committee of Severance Hospital of Yonsei University, Seoul, Republic of Korea, following informed consent (approval no. 4-2019-0060). For surface marker analysis, cultured MSCs were harvested as single cells and incubated with fluorescein isothiocyanate (FITC) or phycoerythrin- (PE-) conjugated antibodies (BD Biosciences Pharmingen, San Diego, CA, USA). The antibodies used in this study were anti-CD14, anti-CD29, anti-CD31, anti-CD34, anti-CD44, anti-CD45, anti-CD73, anti-CD90, anti-CD105, and anti-CD106. Data were acquired using a flow cytometer (Cytomics Flow Cytometer, Beckman Coulter, Fullerton, CA, USA). FITC- and PE-conjugated isotype control antibodies were used as negative controls.