Ab156756

Adipose tissues were collected from the inguinal region of mice and washed completely with PBS to clear the blood. In brief, the tissues were dissected with a sterile surgical knife, and then digested for 30 min in PBS solution containing 2% bovine serum albumin (BSA) and type I collagenase (2 mg/ml). The collagenase was inactivated via DMEM/F12 supplemented with 10% fetal bovine serum (FBS). The procedure was centrifuged with the centrifugation of cell suspension (1200 rpm for 5 min. The isolated cells were collected and cultured in DMEM/F12 with FBS and 1% Penicillin/Streptomycin. After 24 h, the floating cells were removed, and the adherent cells were sub-cultured at 70–80% confluence. For flow cytometric characterization, cells at passage 3 were trypsinized and washed twice with 1% PBS/BSA. Then, cells were incubated with CD105 (ab156756, Abcam), CD90 (sc-53116, Santa Cruz Biotechnology), and CD45 (ab10558, Abcam) for one hour at room temperature and washed in PBS. For labeling, FITC-conjugated secondary antibody was added and samples were kept in the dark for one hour. Samples were fixed in a 1% paraformaldehyde (PFA) solution. Using FACSCalibur® system and FlowJo software (version 7.6.1) cells were analyzed.

The expression levels of the CSps surface markers CD31 (1:200, GB11063-3, Servicebio), CD34 (1:200, ab81289, Abcam), CD90 (1:200, ab225, Abcam), CD105 (1:200, ab156756, Abcam), Sca-1 (1:200, ab51317, Abcam), and KDR (1:200, sc6251, Santa Cruz) were determined by flow cytometry. After a 3-day cultivation, cells and CSps from different substrates were obtained and digested into single cells. After incubation with the primary antibodies for 1 h and the corresponding secondary antibodies for 30 min, Alexa Fluor 488 goat anti-mouse IgG (1:200, ab150113, Abcam) or Alexa Fluor 488 goat anti-rabbit IgG (1:200, ab150077, Abcam) was used. The staining results were analyzed by flow cytometry (BD FACSCanto), and a negative isotypic control was used during the analysis.