A tube formation assay was performed to evaluate the BM-EPC function as described before (Yu et al., 2016 (link)). Briefly, a 96-well plate (Corning, Tewksbury, MA, United States) was coated with 50 μL/well of growth factor–reduced matrix gel (BD Biosciences, San Diego, CA, United States; cat. no. 356231) for 1 h. The cells were then placed on the Matrigel-coated plate with 5 × 105/ml concentration and maintained at 37°C with 5% CO2. After 8 h of incubation, images of the forming tubes were acquired under ×50 magnification using a light microscope. The number of tubes was calculated.
The adhesion ability assay was used to assess the EPC function as previously described (Han et al., 2017a (link)). A total of 5 × 104 BM-EPCs were placed on the mouse vitronectin (1 μg/ml) coated 96-well plate per well. After 2 h incubation at 37°C with 5% CO2, nonadherent cells were softly removed by phosphate-buffered saline (PBS). Then, adherent cells were fixed with 2% paraformaldehyde for 15 min at RT and stained by Hoechst 33258 (10 μg/ml; Beyotime, Shanghai, China; cat. no. C1011). The stained cells (blue color) were observed using a fluorescence microscope (Leica, Wetzlar, Germany) at a magnification of ×400. Each well was counted in 3 random fields.
The adhesion ability assay was used to assess the EPC function as previously described (Han et al., 2017a (link)). A total of 5 × 104 BM-EPCs were placed on the mouse vitronectin (1 μg/ml) coated 96-well plate per well. After 2 h incubation at 37°C with 5% CO2, nonadherent cells were softly removed by phosphate-buffered saline (PBS). Then, adherent cells were fixed with 2% paraformaldehyde for 15 min at RT and stained by Hoechst 33258 (10 μg/ml; Beyotime, Shanghai, China; cat. no. C1011). The stained cells (blue color) were observed using a fluorescence microscope (Leica, Wetzlar, Germany) at a magnification of ×400. Each well was counted in 3 random fields.
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