Pab18289

The differentiated rEPCs were seeded on 25-mm glass-bottom dishes and then immersed in EGM-2 medium overnight. Before immunostaining, the cells were fixed with 4% paraformaldehyde for 15 min and permeabilized in 0.5% Triton X-100 for 20 min at room temperature. Afterwards, the cells were incubated with primary antibody at 4 °C overnight. The primary antibodies included CD34 (PAB18289, Abnova, Taipei, Taiwan), CD133 (PL030395R, PL Laboratories, British Columbia, Canada), VWF (ab6994, Abcam, Cambridge, UK), and IgG (ab6730, Abcam, Cambridge, UK). The cells incubated with IgG served as the negative control. Subsequently, the cells were incubated with FITC fluorescent secondary antibody (bs-0295G-FITC, Biosynthesis Biotechnology Co., Ltd. Beijing, China) at 37 °C for 1 h. Thereafter, the cells were incubated with DAPI (Beyotime Institute of Biotechnology, Shanghai, China) and visualized by fluorescence microscopy (AXIO SCOPE A1, Zeiss, Germany).

Immunophenotyping of the cells for surface and intracellular markers was performed upon reaching 80% confluence of the cultures. The harvested cells were centrifuged at 800g for 10 min, the supernatant was discarded, and then the cells were fixed in 2% paraformaldehyde for 15 min at room temperature (RT), diluted with 5 ml of PBS, and centrifuged at 1,500g for 10 min. The pellet was then resuspended in 1 ml of PBS. For immunostaining, 1 × 10⁵ fixed cells were incubated in 100 μl of Rinsing Solution (Miltenyi Biotec, Somerville, MA, USA) with primary antibodies to CD90 (ab225, 1/100, Abcam), CD45 (130-107-846, clone REA504, 1/20, Miltenyi Biotec, Somerville, MA, USA), CD105 (ab107595, 1/100, Abcam), and CD34 (PAB18289, 1/100, Abnova) at RT for 1 h. Subsequently, the cells were incubated with secondary antibodies: anti-mouse Ig-FITC (ab6785, 1/500, Abcam) or anti-rabbit Ig-PE (sc-3739, 1/100, Santa Cruz) at RT for 1 h in the dark. After the incubation, the cells were washed in PBS, resuspended in 0.5 ml PBS, and then transferred to fresh tubes for analysis using a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) with CellQuest software.