2.4. Monocyte–Endothelial Cell Adhesion Assay

We performed the monocyte–endothelial cell adhesion assay as previously described [20]. We seeded the HUVECs at 1.5 × 10⁵ cells/mL into 96-well plates and incubated overnight to reach confluent monolayers. PLE and each compound were prepared with dimethyl sulfoxide (DMSO) as a stock solution. DMSO was not used at a final concentration of >0.1% (v/v) and was stored at −20 °C until use. We treated the HUVECs with 25, 50, and 100 μg/mL of PLE or compounds (50 μM) for 1 h and then stimulated them with 10 ng/mL TNF-α for 5 h. We prepared the fluorescently labeled THP-1 cells to be added to the activated HUVECs using 2 μM calcein AM (Sigma-Aldrich, St. Louis, MO, USA) for 15 min at 37 °C in phosphate-buffered saline (PBS). After adding the THP-1 cells to the HUVECs, we incubated these for 1 h, washing out the unbound THP-1 cells with PBS three times and measuring the monocyte adhesion at Ex = 485 and Em = 538 nm using a fluorescent plate reader (SpectraMax; Molecular Devices Corporation, Sunnyvale, CA, USA). Parthenolide was used as a positive control because it is a well-known agent to assess the anti-inflammatory effect via inhibition of NF-ĸB activation.