Ldl dylight 550

For LDL uptake cell-based assay, the cells were treated with LDL-DyLight^TM 550 (Cayman chemical, Ann Arbor, Michigan, USA) working solution in serum-free medium. Incubate the cells at 37 °C with 5% CO₂ for an additional 18 hours. At the end of LDL uptake incubation, the culture medium was replaced with fresh culture medium. The degree of LDL uptake was examined under a Leica TCS SP8 confocal microscope (Leica, Wetzlar, Germany) with filters capable of measuring excitation and emission wavelengths 540 and 570 nm, respectively.
For LDL receptors examination, the cells were washed with TBS briefly, and fixed with cell-based assay fixative solution (Cayman chemical) for 10 minutes. Wash the cells with TBST three times for five minutes each. Incubate the cells for one hour with rabbit anti-LDL receptor antibody (Cayman chemical). Wash the cells with TBST three times for five minutes each. Incubate the cells for one hour with Dylight^TM 488-conjugated secondry antibody (Cayman chemical). Wash the cells with TBST three times for five minutes each. The nuclei were counter-stained with DAPI (Sigma-Aldrich). The stained cells were examined under a Leica TCS SP8 confocal microscope (Leica, Wetzlar, Germany).

Huh7 cells were seeded in 6-well tray (3 × 10⁵ cells/well in a complete medium) and after 24 h, treated in MEM/0.4% FBS media. A total of 24 h after treatment, cells were washed with PBS and incubated with 10 µg/mL of LDL-DyLight^TM 550 (Cayman Chemicals cod. 10011229) in 0.4% FCS media. After 3 h of incubation at 37 °C, cells were washed with PBS, detached with trypsin, and resuspended in MEM/10% FBS. After centrifugation (4 min at 3500 rpm), the pellet is resuspended in PBS and each sample was transferred to a cytofluorometer tube. The fluorescence was measured by using a flow cytometer (BD FACSAria™ III, DB Life Sciences, San Jose, CA, 95131, USA) at excitation and emission wavelength of 484 nm.

HepG2 cells were cultured in 96-well plates with 1.5 × 10⁴ cells/well. Then, the cells were treated with 0 (control), 100, and 200 μM of 6-GN for 24 h, and the culture media was removed and replaced with 100 μl/well LDL-DyLight^TM 550 (Cayman) working solution diluted 1:1000 in serum-free culture medium. After incubation for 6 h at 37°C, the culture medium was removed and replaced with fresh culture medium. The degree of LDL uptake was examined under a microscope (Olympus) with filters capable of measuring excitation and emission wavelength 540 and 570 nm, respectively. Subsequently, the cells were ready for the immunocytochemical staining of LDL receptors. After 10 min incubation with 100 μl/well of cell-based assay fixative solution, the cells were washed by TBST three times for 5 min each. Next, the cells were incubated with 100 μl/well of cell-based assay blocking solution for 30 min, and with 100 μl/well of diluted Rabbit Anti-LDL Receptor Primary Antibody for another 1 h. After washed with TBST three times for 5 min each, the cells were incubated in the dark for 1 h with 100 μl/well of diluted DyLight^TM 488-Conjugated Secondary Antibody. At last, the cells were washed with TBST three times for 5 min each, and examined using a fluorescence microscope with a filter capable of excitation and emission at 485 and 535 nm, respectively.