2.6. LDLR Expression and Activity (Lipoprotein Binding and Internalization) by FACS

LDLR cell surface expression was measured by FACS using mouse anti-human-LDLR (1:100; Progen Biotechnik, Heidelberg, Germany) as primary antibody and Alexa Fluor 488-conjugated goat anti-mouse IgG (1:200; Molecular Probes, Eugene, OR, USA) as the secondary antibody. Briefly, cells were incubated at 4 °C, overnight with the primary antibody after fixing (10 min in 4% paraformaldehyde) and blocking (1 h with PBS-5% FBS) steps. Next, cells were incubated for 1 h at room temperature with the secondary antibody. For each sample, the fluorescence of 15,000 events was acquired for data analysis, and measurements were performed in triplicate. All washes were done with PBS-1% BSA.

For quantification of LDLR activity, cells were seeded in 24-well culture plates and transfected, as previously described. Briefly, 24 h after transfection, cells were incubated for 3 h at 37 °C or 4 °C with 20 μg/mL FITC-LDL to determine LDL internalization or LDL-LDLR binding, respectively. After incubation, cells were washed three times, fixed for 10 min in 4% paraformaldehyde, and washed again three times. The amount of internalized LDL was determined by adding Trypan blue solution to a 0.2% final concentration (Sigma-Aldrich, Steinheim, Germany) to the samples. This dye quenches external fluorescence, eliminating the signal of non-internalized LDLR-LDL complexes, allowing the determination of the intensity of the remaining fluorescent particles inside the cells. Fluorescence intensities were measured by flow cytometry in a Facscalibur Flow cytometer. For each sample, the fluorescence of 15,000 events was acquired for data analysis, and measurements were performed in triplicate. All washes were done with PBS-1% BSA.