Recombinant human pcsk9

Cultured CaCo-2 cells were obtained from American Type Culture Collection (ATCC, VA). CaCo-2 cells were first grown for 3 days in EMEM specialty medium (ATCC, VA) in 20% FBS supplemented with 1% penicillin-streptomycin, at 37°C, 5% CO₂, until they reached 80–90% confluence. Induction of cell polarization is necessary for CaCo-2 cells to resemble human intestinal enterocytes in vivo. To induce polarization, CaCo-2 cells were maintained in culture in 10% FBS-DMEM for 14 days in specialized, collagen-treated transwell plates with 0.4 μM pore size polycarbonate filters (CoStar, DC).
Unless otherwise indicated, all studies were performed at least three times. Optimally polarized confluent CaCo-2 cells in 1% FBS-DMEM were treated for 24 hours with recombinant human PCSK9 (R&D Systems, MN) reconstituted in Millipore H₂0. PCSK9 was mostly used at the concentration of 10 μg/mL for 24 hours^{13 (link)}, although many experiments included different doses (0, 5, 7.5, 10 and 12 μg/mL) and different times (0, 2, 4, 8, 12, and 24 hours).