L3224

After 3 days of culture, the media was replaced with a live/dead staining solution containing 2 μM Calcein-AM and 4 μM Ethidium homodimer-1 (Etd1) in the culture media (Thermo Fisher cat#L3224) and incubated for 30 min in a culture incubator. In the final 8 min, Hoechst 33342 (Thermo Fisher cat# H3570) was added at a concentration of 5 µg/mL to each sample. Images were collected on an EVOS imaging system. Images were analyzed for Calcein-AM positivity to identify the number of cellular structures. Each structure was designated as either live (Calcein+) or dead (Ethd2+). Higher magnifications were taken to analyze the nuclear structure stained with Hoechst 33342. Comparisons between culture conditions were done via z-test.

For each test, ethidium homodimer-1 (2 μM) (L3224, Thermo Fisher Scientific, Waltham, MA, United States) and calcein-AM (4 μM) were added to PBS and mixed. Afterwards, the plate was incubated for 30 min and rinsed with PBS three times. The cells were observed under fluorescence microscopy.

For the lactate dehydrogenase (LDH) (Pierce, Thermo Fisher Scientific) assay, the positive control was a sample medium collected from cells that were lysed with a buffer reagent provided by the manufacturer. The negative control was medium from cells cultured on a tissue culture plate under standard culture conditions. Supernatant from unstimulated and stimulated cells in 2D and 3D PPy scaffolds were also collected. Supernatant from all conditions was then mixed with the reaction mixture and later added with the stop solution. The LDH activity was measured by the Spectra Max M2 plate reader (Molecular Devices) at an absorbance of 490 nm and 680 nm, based on the manufacturer’s protocol. For the alamar blue assay, the positive control was cells cultured on a tissue culture plate under standard culture conditions, whereas the negative control was lysed cells. A 10% alamar blue reagent (DAL1025, Thermo Fisher Scientific) was added to each culture condition including the unstimulated and stimulated cells in the 2D and 3D PPy scaffolds. The activity from the alamar blue assay was quantified with the plate reader by monitoring the absorbance of the reagent at 570 nm while using 600 nm as a reference wavelength based on the manufacturer’s protocol. Live/Dead assay was used to stain cells based on the manufacturer protocol (L3224, Thermo Fisher Scientific).

After culturing EPCs with conditioned medium for 2 days, RT-qPCR was used to measure the expression level of angiogenic genes [von Willebrand factor (vWF), endothelial nitric oxide synthase (eNOS), fibroblast growth factors (FGF), vascular endothelial growth factor-A (VEGFA)]. At the same time, cells cultured with α-MEM were used as a control group. To intuitively understand the effect of BMSCs on angiogenesis, we cultured EPCs grown on Matrigel with conditioned medium and observed tube formation at different times. Generally, the 100 μl of Matrigel (Corning, USA) was placed into a 48-well plate, then 2 × 10⁴ cells were added to each well including 1 ml of conditioned medium with α-MEM as the control. Angiogenesis status was imaged under a microscope (Leica) after 4 h and 6 h of incubation. After 6 h of culture, the cells were stained with a live-dead kit (L3224, Thermo Fisher) and observed under a fluorescence microscope (EVOS FL Auto, Life Technologies, USA). The total length, number of segments and nodes in 6 randomly chosen fields were quantified using the Angiogenesis Analyzer macro in ImageJ [40 (link)].