Spot pursuit camera

For immunocytochemistry, cells were washed once with PBS and fixed in 4% paraformaldehyde (PFA) for 15 min. Cells were then blocked with 10% normal donkey serum in PBS (blocking buffer) for 1 h. When probing for an intracellular antigen, 0.3% Triton‐X was included in the blocking buffer. Cells were then incubated with the primary antibodies lamin A/C (MAB3211; Millipore, 1:250), progerin (Cao et al., 2011 (link)) with a dilution of 1: 250, goat anti‐VE‐cadherin (AF938; R&D Systems, 1:100), and sheep anti‐PECAM‐1 (AF806, R&D Systems, 1:100), in 5% normal donkey serum in PBS overnight at 4°C. After three washes with PBS, cells were incubated in 1% BSA in PBS‐containing secondary antibodies and DAPI (Vector Laboratories). Secondary antibodies used for immunocytochemistry were Alexa Fluor^® 594 donkey anti‐rabbit IgG (Invitrogen, 1:1000) and Alexa Fluor^® 488 donkey anti‐mouse IgG (Invitrogen, 1:1000 dilution). Cells were washed three times with PBS, and images were acquired with Zeiss AX10 microscope equipped with a SPOT PURSUIT camera. For the analysis of nuclear morphology, greater than 120 nuclei were analyzed per condition where the nuclei were assigned by visual inspection into normal or abnormal nuclear phenotype.

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.