Mesenchymal stem cells (MSCs) were derived from the adipose tissue of male transgenic Sprague-Dawley (SD) rats expressing Green Fluorescent Protein (GFP) (SD-Tg(CAG-EGFP)CZ-0040sb) (SLC Inc., Japan). Briefly, the tissue was washed extensively with sterile phosphate buffered saline (PBS) and then the tissue was treated with 0.1% collagenase (type I; Sigma-Aldrich) in PBS for 30 minutes at 37 °C with gentle agitation. After filtration through 100-μm mesh filter to remove debris, the filtrate was washed three times and completely suspended in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine. The cultures were maintained in an incubator with a humidified atmosphere of 5% CO2.
The cell phenotype of MSCs was tested by flow cytometer with a FACScan argon laser (BD Bioscience, San Jose, CA). MSC suspension was washed and stained with phycoerythrin-conjugated antibodies (red fluorescence) against CD45 and CD90 (Abcam Inc., Cambridge, UK) and CD 29 (Biolegend, San Diego, CA). Isotype-matched negative controls were used to assess background fluorescence.
The cell phenotype of MSCs was tested by flow cytometer with a FACScan argon laser (BD Bioscience, San Jose, CA). MSC suspension was washed and stained with phycoerythrin-conjugated antibodies (red fluorescence) against CD45 and CD90 (Abcam Inc., Cambridge, UK) and CD 29 (Biolegend, San Diego, CA). Isotype-matched negative controls were used to assess background fluorescence.