4.7. Nitric oxide assays

After 48 h of incubation, 1 ml media was taken and flash‐frozen in liquid nitrogen and frozen at −80°C. For the cell in the fluidic bioreactors, after 24 h of perfusion, 1 ml media was removed from the flow circuit and flash‐frozen in liquid nitrogen and frozen at −80°C. Prior to testing, samples were spun at 9500 RCF for 7 min with a 10,000 MWCO spin column (Corning). Total nitrate and nitrite were measured using a colorimetric assay (Arbor Assays) as per kit instructions. Absorbance was measured at 540 nm using a 96‐well microplate reader. For the second method of intracellular NO level measurement, we used DAF‐FM (Thermo Scientific). iPSC‐ECs were either treated with 0, 0.25, 0.5, and 1 mM nitric oxide donor S‐Nitroso‐N‐acetyl‐DL‐penicillamine (SNAP, N3398; Sigma‐Aldrich), or with an eNOS inhibitor (0.2 mM N‐omega‐nitro‐L‐arginine methyl ester hydrochloride; L‐NAME, N5751; Sigma‐Aldrich), and then incubated with DAF‐FM 10 μM at 37°C, and 5% CO₂ for 30 min as per manufacturer's recommendation. Excess probe was removed by washing with PBS, and the cells were switched to fresh media prior to imaging. Images were acquired with Zeiss AX10 microscope equipped with a SPOT PURSUIT camera. The intensity of the DAF‐FM fluorescent signal (intracellular NO level) was measured using ImageJ and plotted as mean of the fluorescent signal (integrated density) in each cell.