Bodipy ldl

HepG2 assay for measuring LDL uptake HepG2 cells were purchased from ATCC, authenticated by Short Tandem Repeat profiling and tested negative for mycoplasma. HepG2 cells were seeded at a density of 8,000 cells in 40 μL per well in black 384-well Greiner μClear plates in EMEM (ATCC) containing 10% Fetal Bovine Serum (Cytiva) and incubated for 24 hours at 37°C, 5% CO 2 . The next morning, cells were washed once with, and incubated in, EMEM containing 2% Human Lipoprotein Deficient Serum (Intracel, Sigma) for 24 hour at 37°C, 5% CO 2 . Cells were treated with antibody or peptide and PCSK9 mixture (40 μL, pre-incubated for 45 minutes at room temperature) for 1 hour at 37°C, 5% CO 2 . Following the incubation, 10 μL of Bodipy-LDL was added (Thermo Fisher) to the assay mixture and incubated for 2 hours at 37°C, 5% CO 2 . Twenty-five μL of 12% paraformaldehyde containing 1.5 μg/mL Hoechst 33342 was added to each well, and the cell plates were left at room temperature for 20 minutes. Medium was aspirated and cells washed twice with PBS, 50 μL PBS containing 0.03% sodium azide was added to each well and sealed. Cell plates were scanned, and images were obtained with an INCELL Analyzer. The compounds were evaluated in a minimum of 3 separate (11-point titration) experiments and n of 3 per concentration.