Aortic Valve Tissue Preparation and Analysis

Aortic valves were preserved and transported in PBS, and processed within a maximum of 2 h after extraction. The valves were washed immediately in PBS to reduce blood contaminants. For 2D-DIGE and western blotting, one aortic valve leaflet was ground into a powder in liquid N₂ in a mortar. Protein extracts were then prepared from the valve as described previously [8 (link), 9 (link)] and the total protein concentration was measured by the Bradford-Lowry method (Bio-Rad protein assay) [10 ].
For secretome analysis, the second valve leaaflet was cultured as described elsewhere [11 (link), 12 (link)] using medium supplemented with antibiotics and amphotericin B (Fungizone^®) to avoid contamination. Samples were transferred to a Petri dish (Cell Star^®), cut into pieces and incubated at 37 °C in lysine-arginine free 1640 RPMI medium (Cell Culture Technologies Invitrus) supplemented with 5 mg/ml fungizone, 250 mg/ml amikacin, 2 mg/ml l-lysine 2HCl (U-13C6, 97–99%) and 10 mg/ml l-Arginine HCl (U-13C6, 97–98%: Cambridge Isotope Laboratories Inc., Andover, MA) in a humidified atmosphere of 5% CO₂. The valves were cultured for 96 h and the medium collected was stored at −80 °C until analysis. Finally, the valves were fixed in formalin at 4 °C, decalcified in Shandon-TBD1 (Thermo Scientific) and embedded in OCT for subsequent immunohistochemistry.