ADMSCs were isolated from inguinal fat pad of Sprague–Dawley rats (Slack Experimental Animal Company, Shanghai, China) based on a published method.20 (link) Briefly, the lipoaspirate was minced and digested with 1% collagenase A (Gibco, Carlsbad, CA) and washed with phosphate-buffered saline (PBS). After filtration and centrifugation, cells were then resuspended in Dulbecco's modified Eagle's medium (DMEM; Gibco) containing 10% fetal calf serum (FCS; Gibco) and cultured in a humidified 5% CO2 atmosphere at 37°C; the cells in passages 3 and 4 were identified and cultured for future experiments.
The collected ADMSCs were then observed by microscope and analyzed by flow cytometry. About 5 × 105 ADMSCs were resuspended in 1 mL of PBS and incubated at 4°C for 30 minutes with the following antirat antibodies (all from BD Biosciences, San Jose, CA): phycoerythrin-conjugated CD29, CD44, and D-related human leukocyte antigen (HLA-DR); fluorescein isothiocyanate–conjugated CD31, CD45, and CD105. Phycoerythrin-conjugated rat immunoglobulin G1 and fluorescein isothiocyanate–conjugated rat immunoglobulin G1 (BD Biosciences, San Diego, CA) were used as isotype controls. After washing, the specimens were analyzed within 1 hour using an FACSCalibur flow cytometer (BD Biosciences).
The collected ADMSCs were then observed by microscope and analyzed by flow cytometry. About 5 × 105 ADMSCs were resuspended in 1 mL of PBS and incubated at 4°C for 30 minutes with the following antirat antibodies (all from BD Biosciences, San Jose, CA): phycoerythrin-conjugated CD29, CD44, and D-related human leukocyte antigen (HLA-DR); fluorescein isothiocyanate–conjugated CD31, CD45, and CD105. Phycoerythrin-conjugated rat immunoglobulin G1 and fluorescein isothiocyanate–conjugated rat immunoglobulin G1 (BD Biosciences, San Diego, CA) were used as isotype controls. After washing, the specimens were analyzed within 1 hour using an FACSCalibur flow cytometer (BD Biosciences).
