For serum collection, tubes of blood were removed from the ice and left at room temperature for 30 minutes to clot; then, the clots were dislodged using p200 pipette tips. For plasma collection, retroorbital blood draws were performed using microhematocrit capillary tubes (Thermo Fisher Scientific, 22-362566) and collected in heparinized microtainers (BD Biosciences, 365965). Blood samples were spun down in a tabletop centrifuge for 10 minutes at 4°C and 10,000g. The supernatant (serum or plasma) was removed from the tube and transferred to a new Eppendorf for storage at –80°C until analysis.
Serum was analyzed in batches for total cholesterol to confirm that animals in the atherogenic arm of the study responded to the AAV-m-PCSK9 and high-fat/high-cholesterol diet. FujiFilm Wako Diagnostics kits for Cholesterol E (no. 999-02601) were used according to manufacturer recommendations. Samples were diluted down 1:10 in sterile water so they fell on the standard curve. All tests were completed in triplicate.
Serum was analyzed in batches for total cholesterol to confirm that animals in the atherogenic arm of the study responded to the AAV-m-PCSK9 and high-fat/high-cholesterol diet. FujiFilm Wako Diagnostics kits for Cholesterol E (no. 999-02601) were used according to manufacturer recommendations. Samples were diluted down 1:10 in sterile water so they fell on the standard curve. All tests were completed in triplicate.