Ab19622

Porcine DFAT cells at passage 4 (P4) were detected by flow cytometric for cell surface antigen. The cells were digested with 0.25% trypsin-EDTA and incubated with primary antibody CD29 (552369, BD, USA), CD31 (MCA1746PET, AbD Serotec, USA), CD34 (GB12013, China), CD44 (ab19622, Abcam, USA), and CD90 (562245, BD, USA) for 30 min at 4°C and further they were incubated for 30 min at 4°C with FITC or CY3 conjugated secondary antibody. Cell fluorescence was detected by FACSCalibur instrument (Becton Dickinson, USA). The data were analyzed using CellQuest Software (Becton Dickinson, USA).

The DMSCs of passages 3 were subcultured on a 24-well plate, the cells were fixed in 4% (w/v) PFA (paraformaldehyde) for 15 min and then washed with ice-cold PBS three times (5 min each). Cells were permeabilized by 0.25% (v/v) Triton X-100 (Sigma) for 10 min. The cells were then washed three times (5 min per wash) with PBS and incubated with goat serum (Zhongshan Golden Bridge) at room temperature for 30 min. Then we added anti-CD29 (1:100, sc-53711, Santa Cruz) and anti-CD44 (1:100, ab19622, Abcam), and incubated the cells overnight at 4°C. The primary antibody was removed and cells were washed three times (5 min per wash) with PBS. We then added FITC-conjugated goat anti-mouse or FITC-conjugated goat anti-rat antibodies (Zhongshan Golden Bridge) and incubated the cells at room temperature in the dark for 1 h. The plate was washed three times (5 min per wash) with PBS in the dark. Finally, the cells were incubated with 10 μg/ml DAPI (4′,6-diamidino-2-phenylindole) for 15 min and then washed three times with PBS. Images were obtained using a laser-scanning confocal microscope. Ten randomly selected non-overlapping fields of vision were observed and photographed (Nikon).