*4.8. Determination of CD11c Expression in THP-1 Cells Treated with Di*ff*erent Doses of L5 by Flow Cytometry Analysis*

The human monocytic cell line, THP-1 cells (ATCC TIB-202; American Type Culture Collection, Manassas, VA, USA), was grown in RPMI 1640 (Thermo Fisher Scientific, Taichung, Taiwan) supplemented with 10% FBS and 1% penicillin/streptomycin antibiotics in an incubator (Thermo Fisher Scientific GmbH, Dreieich, Germany) containing 5% CO2 at 37 ◦C. At the beginning of the lipid treatment experiment, the medium was changed to RPMI with 1% lipid-depleted FBS. THP-1 cells, at a density of 1 <sup>×</sup> 105 cells/mL, were treated with L5 (10 or 50 <sup>μ</sup>g/mL) at 37 ◦C for 2 days. The cells were harvested and washed with phosphate-buffered saline (PBS) and then blocked with TruStain FcX (BioLegend, San Diego, CA, USA) at room temperature for 10 min. The CD11c levels of THP-1 samples were quantified by using FITC (fluorescein isothiocyanate)-conjugated anti-CD11c antibody (BD Biosciences Pharmingen, San Diego, CA, USA) and flow cytometry FACSCelesta™ (BD Biosciences) according to the manufacturer's protocol and the described technique [45]. FITC-conjugated IgG isotype antibody (BD Biosciences) (4 ◦C, 30 min) served as isotype controls. We analyzed at least 10,000 cells/condition in duplicate. Data analysis was performed with FlowJo software (BD Biosciences). We gain major cell populations (named G1) with dot plots (FSC:SSC) by using an unstained control. Data were expressed as the frequency of CD11c in the gated cell population
