Diaphot 150

HUVECs (passage 4) were grown to confluence in 35 mm, glass-bottom microwell dishes, and handled as described in (Krupp et al., 2013 (link),Boeldt et al., 2015 (link),Boeldt et al., 2017 (link)). HUVECs were incubated with 10uM Fura2-AM (Molecular Probes) with 0.05% Pluronic acid F127 (Life Technologies) in HEH for 1 hour at 37°C. HUVECs were washed with Krebs buffer following incubation and HUVECs were left in Krebs buffer for 30 minutes at room temperature, allowing for complete ester hydrolysis. The dish was then placed on a Nikon inverted microscope (Diaphot 150; Nikon, Melville, NY) for imaging. Up to 99 cells per field of view were identified and manually circled for analysis (Krupp et al., 2013 (link)). After 5 minutes baseline recording, HUVECs were stimulated by 100 uM ATP (ATP exposure 1) and recorded for a total of 30 minutes using alternate excitation at 340 nm and 380 nm at 1 second intervals. Number of cells that had ≥3 bursts were noted and calculated. Cells were then washed three times with Kreb’s buffer for 20 minutes to bring cells to baseline for further imaging steps (A and B below). A standard curve was used to calculate concentration of intracellular Ca²⁺ ([Ca²⁺]ⁱ).

After growing P-UAEC to near confluence on 35-mm glass bottom dishes, media with serum was removed, and cells were loaded with the ROS-sensitive dye H₂DCFDA (5 μM in Krebs buffer) for 30 min and then were washed three times with Krebs buffer. H₂DCFDA is deesterified intracellularly into a non-fluorescent polar derivative (H2DCF), and then is oxidized to its fluorescent form 2′,7′-dichlorofluorescein (DCF) by ROS (Sauer et al., 2000 (link)). Cells were recorded for 5 min to establish baseline before addition of various doses of TNF-alpha with recording continuing for 1 h. Fluorescence of DCF was monitored at 485 nm excitation and 535 emission wavelengths. Images were recorded with a digital camera (PixelFly, Cooke Corp., Romulus, MI) connected to an inverted microscope (Nikon Diaphot 150, Melville, NY). The fluorescent intensities were quantified using InCyt Im1 software (Intracellular Imaging, Inc., Cincinnati, OH) and normalized to the starting fluorescence (F/F₀).