MLECs were collected after digestion with PBS supplemented with 2 mM
EDTA and 1% BSA (PBS-EB), and adjusted at 106 cells/ml. For FGF2
binding, the dissociated cells were incubated with biotinylated FGF2 (20
μg/ml) on ice for 30 min. Following, the cells were incubated with
eFluor®450-conjugated streptavidin and were then subjected
to flow cytometry analysis (LSRII, BD Biosciences). For endothelial cell marker
analysis, the cells were incubated with FITC-conjugated anti-mouse CD31
(eBioscience) or PE-conjugated anti-mouse VEGFR2 antibody (BD Pharmingen) on ice
for 30 min and were then subjected to flow cytometry analysis. For HS phage
display antibody staining, 106 MLECs in 100 μl PBS-EB were
firstly incubated with 10 μl antibody (AO4B08, HS4C3, RB4EA12, and EV3C3)
on ice for 1 h and then incubated with biotinylated anti-VSV-G antibody (Abcam)
on ice for 1 h after washing. After further incubating with
streptavidin-conjugated eFluor®450 (eBioscience) and propidium
iodide (25 μg/ml) for 30 min on ice, the cells were subjected to flow
cytometry analysis. The flow cytometry data was processed and analyzed using
FlowJo software.
EDTA and 1% BSA (PBS-EB), and adjusted at 106 cells/ml. For FGF2
binding, the dissociated cells were incubated with biotinylated FGF2 (20
μg/ml) on ice for 30 min. Following, the cells were incubated with
eFluor®450-conjugated streptavidin and were then subjected
to flow cytometry analysis (LSRII, BD Biosciences). For endothelial cell marker
analysis, the cells were incubated with FITC-conjugated anti-mouse CD31
(eBioscience) or PE-conjugated anti-mouse VEGFR2 antibody (BD Pharmingen) on ice
for 30 min and were then subjected to flow cytometry analysis. For HS phage
display antibody staining, 106 MLECs in 100 μl PBS-EB were
firstly incubated with 10 μl antibody (AO4B08, HS4C3, RB4EA12, and EV3C3)
on ice for 1 h and then incubated with biotinylated anti-VSV-G antibody (Abcam)
on ice for 1 h after washing. After further incubating with
streptavidin-conjugated eFluor®450 (eBioscience) and propidium
iodide (25 μg/ml) for 30 min on ice, the cells were subjected to flow
cytometry analysis. The flow cytometry data was processed and analyzed using
FlowJo software.