The bioactivity of POCR was assessed using HUVECs and HASMCs (Lonza, Walkersville, MD) that were within passage 4–7. To assess cell toxicity, cells were seeded in the pretreated 96-well plates at a seeding density of 5000 cells/cm2. After 48 h of culture, the LIVE/DEAD viability kit (Life Technologies, Carlsbad, CA) was used according to the manufacturer’s instructions to measure the ethidium homodimer and calcein staining of dead and live cells, respectively.
For cell proliferation measurements, HUVECs and HASMCs were seeded in the pretreated 96-well plates. HUVECs were seeded at 3000 cells/cm2, and HaSMCs were seeded at 5000 cells/cm2. Cell lysates were collected at 1, 3, and 5 days for measurement of DNA content using the PicoGreen dsDNA quantitation reagent (Life Technologies, Carlsbad, CA). Briefly, cells were rinsed with PBS three times, and 100 µL of cell lysis buffer containing 1% Triton X-100 were added to each well. After 15 min of incubation at room temperature, 50 µL of cell lysate was collected from each well. Cell lysates were mixed with PicoGreen at a 1:1 volume ratio. To measure the cell number, a standard curve was generated using known cell numbers in culture and their corresponding fluorescence intensity.
For cell migration measurements, a scratch migration test was performed. HUVECs and HASMCs were seeded in pretreated 24-well plates. Upon reaching 100% confluence, cells were serum-starved with basal media for 12 h (HUVECs with EBM-2, HASMCs with SmBM-2). Subsequently, a vertical scratch was made in each well, cells washed with prewarmed PBS and cells cultured for another 24 h in basal media. The remaining scratched area was measured using ImageJ software to determine the in vitro effect on cell migration. Full paper
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