Collagenase 2 solution

VICs were harvested from aortic valve leaflets of fresh porcine hearts obtained from a local commercial abattoir (Fisher Ham and Meats, Spring, TX) according to a validated protocol.³¹ To loosen endothelial cells, dissected leaflets were digested in a collagenase 2 solution (500 U/mL, Worthington Biochemical, Lakewood, NJ) for 30 min at 37 °C. After the endothelial cells were scraped from both surfaces of the leaflets, the residual leaflet tissue was minced. The minced tissue was digested in a collagenase 3 solution (300 U/mL, Worthington Biochemical) for 4 h at 37 °C. The solution was passed through a 70 µm cell strainer and then VICs were plated in tissue culture polystyrene (TCPS) flasks. VICs were cultured in a standard humidified incubator (37 °C, 5% CO₂) in 50:50 DMEM/F12 media (Corning, Tewksbury, MA) with 10% bovine growth serum (BGS, Lonza, Walkerville, MD), 1% 1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, Thermo Fisher Scientific, Waltham, MA), and 1% penicillin/streptomycin/ amphotericin (Corning). For consistency, all VICs were frozen after passage 1 and used in passages 2–3. VICs were used from three separate harvests with cells pooled from all aortic valve leaflets of six porcine hearts during each harvest.

Visceral fat deposits and a 5cm section of the ileum were harvested from mice and washed with PBS before being placed in a 0.2% (2mg/mL) collagenase 2 solution, (Worthington Biochemical; Lakewood, NJ, USA) diluted in RIPA buffer (Gibco, ThermoFisher Scientific; Waltham, MA, USA), under contestant stirring for 60 minutes at 37°C. The isolated cells were filtered through a 70 μM cell strainer and red blood cells were lysed with ACK lysis buffer (VWR; Radnor, PA, USA). Cells were then stained as described above for the bone marrow.