Growth factor reduced matrix gel

Matrigel tube formation assay was used to evaluate the protective role of PCB2 on EPC angiogenic function under HG. Briefly, 96‐well plates were coated with growth factor‐reduced matrix gel (50 μL/well, BD Biosciences). EPCs (2 x 10⁴ cells/well) were suspended in 100 µL basal culture medium containing HG or the same dose of mannitol in the absence or presence of different concentrations of PCB2 (0.1 μmol/L, 0.5 μmol/L, 2.5 μmol/L) and seeded on the 96‐well pre‐coated with matrix gel. Tube structure formed after EPCs incubation at 37°C with 5% CO₂ for 6 hours and microscopic images were acquired using an inverted microscopy (Nikon Eclipse E600, Nikon). The total length of tube structures in each well was calculated by Image J software.

The in vitro angiogenic capability of EPCs was determined by a Matrigel tube formation assay. Briefly, 48‐well plates were coated with growth factor‐reduced matrix gel (150 μl/well, BD Biosciences). EPCs (5 × 10⁴ cells/well) in 200‐μl basal culture medium or medium containing HG in the presence or absence of different concentrations of sitagliptin were incubated at 37°C with 5% CO₂ for 12 hrs to form tubes. In some experiments, cells were pretreated with 3‐MA (5 mmol/l) or rapamycin (10 μmol/l) for 30 min. and then continually exposed to HG for the indicated durations in the presence or absence of sitagliptin. Images of tubes in each well were acquired using an inverted microscope (Nikon Eclipse E600, Nikon, Kanagawa, Japan). The tube lengths were calculated by ImageJ software (National Institutes of Health, Bethesda, MD, USA).

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.