*4.6. Examination of Foam Cells Formation in Monocyte-Derived Macrophages Treated with L1 or L5*

To induce differentiation of human monocytes into macrophages, THP-1 cells (1 <sup>×</sup> 105 cells/mL) were grown in media and treated with 10 ng/mL PMA (MilliporeSigma, Temecula, CA, USA) for 48 h. The culture medium was subsequently changed to RPMI and 1% lipid-depleted fetal bovine serum (FBS) from differential ultracentrifugation as described previously [43]. Then, the macrophages were treated with different doses of L1 or L5 (10, 25, or 50 μg/mL) at 37 ◦C for 48 h. Foam cell formation in macrophages was examined by using Oil red O staining [44]. The images for foam cell formation were observed in an ECLIPSE 50i microscope (Nikon, Tokyo, Japan) and captured at 100× with NIS-Elements software (Nikon, Tokyo, Japan). The percentage of foam cell formation was quantitated by dividing the number of Oil red O staining macrophages by the total number of macrophages in 2 random microscopic fields.
