Endothelial cell and fibroblast lysate/cell membrane fractions

Cell lysates (containing solubilized cell membranes) were prepared from HUVECs, GMVECs, LSECs, and fibroblasts grown in T-75 and T-25 flasks until confluence. Cells were washed 3X with Ca⁺²/Mg⁺²-containing PBS, and then the media was replaced with serum-free medium (MCDB-131 + insulin [10 mg/l]-transferrin [5.5 mg/ l]-selenium [6.7 µg/ l], Life Technologies) containing 5 mM CaCl₂ for 24 h (500 µl per T-25 flask and 1.5 ml per T-75 flask). On collection day, cells were washed 3X with 10 ml (T-75) or 4 ml (T-25) of cold, sterile Tris, pH 7.3 buffer (50 mM Tris, 1% BSA, and 5 mM CaCl₂) to remove any exogenous proteins. Cells in T-75 flasks were lysed with 500 µl of CelLytic M (ice cold, Sigma-Aldrich C-2978) + 10 µl of Halt protease/phosphatase inhibitor cocktail (Thermo Scientific, 78,430) for 15 min with rocking. T-25 flasks required 175 µl of CelLytic M + 3 µl of inhibitor cocktail. Lysates used for thrombin activity measurement were prepared without the inhibitor cocktail to preserve thrombin activity. Lysed cells were collected with cell scrapers, placed into chilled tubes, and centrifuged at 12,000 g for 15 min at 4 °C. The soluble fractions were collected and stored at − 80 °C until used for specific protein quantification assays.