Fluoview 500 confocal system

DTAs were dissected from multiple 8–18-week-old male and female wild-type and smLRP1^−/− mice. mAoSMCs were isolated as described above from individual DTAs and cultured in Vascular Smooth Muscle Cell Growth Medium (ATCC^®), 10% FBS on 0.1% gelatin (Sigma-Aldrich) coated 35 mm glass bottom culture dishes (MatTek Corporation). Cultures were maintained at 37°C, 5% CO₂ in a humidified atmosphere until imaging by confocal microscopy at passage 1–2. Growth medium was aspirated from each dish and mAoSMCs were incubated in Krebs buffer with 1 μM rhod-2-AM (Life Technologies) for 30 minutes at room temperature protected from light. After 30 minutes, the buffer-dye solution was aspirated from the culture dish and dye loaded mAoSMCs were incubated in Krebs buffer for an additional 30 minutes at room temperature protected from light. Resting steady-state fluorescence levels (F₀) were imaged and recorded for 3–9 cells per dish at room temperature using a Fluoview 500 confocal system mounted on an Olympus IX71 inverted microscope and viewed with a 60×/1.20 NA water immersion objective. Fibers were excited at 532 nm, and the fluorescence emitted above 550 nm was detected. Images were analyzed as detailed in the online-only Data Supplement.