Rat anti mouse cd44

The cell surface markers of MSCs were analyzed using a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA). Briefly, cells that reached 90% confluence were harvested using 0.25% EDTA and washed twice in Dulbecco’s phosphate buffered saline supplemented with 10% FBS. The cells for detecting CD11b, CD34, CD45, Sca-1, CD44 and CD73 were labeled directly with BB515 or PE-conjugated CD markers (rat anti-mouse CD11b [1: 100, BD Pharmingen; BD Biosciences, Franklin Lake, NJ, USA], rat anti-mouse CD34 [1: 100, BD Pharmingen], rat anti-mouse CD45 [1: 100, BD Pharmingen], rat anti-mouse Sca-1 [1: 100, BD Pharmingen], rat anti-mouse CD44 [1: 100, BD Pharmingen], rat anti-mouse CD73 [1: 100, BD Pharmingen]).

ADSCs were isolated from the inguinal adipose tissue of ten 8-week-old male C57BL/6N mice according to the method described by Nakagami et al. [4 (link)] with minor modifications. The inguinal adipose tissue samples were treated with 0.1% collagenase and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin mixture. We used ADSCs at passage 5 in this experiment. Similarly, ADSCs were isolated from ten 8-week-old male C57BL/6-Tg (CAG-EGFP) mice to evaluate the engraftment rate of these cells. The identity of ADSCs was confirmed through the evaluation of various cell surface markers using flow cytometry. ADSCs were mechanically dissociated and resuspended in fluorescence-activated cell sorting (FACS) staining buffer (phosphate-buffered saline supplemented with 5% fetal bovine serum). The antibodies used for FACS analysis included rat anti-mouse CD44, CD73, CD90, and Sca-1 (BD Biosciences, San Jose, CA, USA) conjugated to fluorescein isothiocyanate or phycoerythrin. The cells were stained for 30 min at room temperature, washed, and examined using the BD FACSVerse™ Flow Cytometer (BD Biosciences). The data were analyzed using BD FACSDiva™ (BD Biosciences).