Recombinant mouse sflt1 protein

Migration activity of cultured EPCs was evaluated with a chemotaxis chamber assay as described previously (12). Briefly, the polycarbonate filter (5-mm pore size) (Transwell; Corning, Corning, NY, USA) was placed between the upper and lower chambers. The cultured EPCs were coincubated with recombinant mouse sFlt1 protein (300 ng/ml; R&D Systems, Minneapolis, MN, USA) for 24 h in 0.5% FBS/endothelial basal medium 2 (EBM2) (Lonza, Tokyo, Japan) before migration assay. The sFlt1-pretreated EPC suspensions ( 5´ 10 4 cells/well) were placed in the upper chamber, and the lower chamber was filled with 0.5% FBS/EBM2 (Lonza) with growth factors alone or that with a chemoattractant SDF-1a (100 ng/ml) (R&D Systems). The cells were incubated for 16 h at 37°C in 5% CO 2 . The total number of migrated cells on the lower chamber side of polycarbonate filter was evaluated in each well and expressed as the migration activity. The polycarbonate filter with migrated cells was fixed with 2% PFA/PBS and stained with 4,6-diamidinophenylindole (DAPI) (Sigma-Aldrich) to visualize nuclei. The DAPI-positive nuclei were counted in three polycarbonate filters and averaged for comparison. The experiment was repeated for over three times, and the representative result was demonstrated.

Proliferation activity and viability of the cultured EPCs were examined with the use of Cell Counting Kit-8 (Dojindo, Tokyo, Japan) according to the manufacturer's instructions (12). Briefly, cultured EPCs (10,000 cells/well) were seeded on 96-well flat-bottomed plates with 100 ml of growth medium (EGM2-MV BulletKit; Lonza) Then cells were incubated in the presence (100 ng/ml) or absence of recombinant mouse sFlt1 protein (R&D Systems) for 48 h at 37°C in 5% CO 2 . The absorbance at 570-nm wavelength was recorded in eight wells in each group with the use of a 96-well enzyme-linked immunosorbent assay (ELISA) plate reader (SPECTRA MAX 190; Molecular Devices, Tokyo, Japan), and the data were averaged for comparison. The experiment was repeated for over three times, and the representative result was demonstrated.