Monocyte-endothelial cell adhesion assay

THP-1 cells (ATCC, TIB-202) were labeled with 2.5 μM calcein AM (Sigma, C1359), or CM-DiI (ThermoFisher Scientific, V22888) in a 37 °C 5% CO₂ incubator for 30 minutes. The labeled cells were washed with 10 ml of PBS for 3 times to remove the residual calcein AM. After the final wash, the cells were gently resuspended at a final concentration of 1×10⁶ cells/ml in culture medium. Calcein AM-labeled THP-1 cells (1 × 10⁶) were added to a confluent monolayer of primary cultured ECs, which were treated with vehicle or 20 ng/ml of TNF-α (Sigma, GF023) for 24 hours. CM-DiI-labeled THP-1 cells (1 × 10⁶) were added to a confluent monolayer of HUVECs, which were transduced with an adenovirus expressing GFP (Ad-GFP), RGC-32 shRNA (Ad-shRGC32), or RGC-32 cDNA (Ad-RGC32). Ad-RGC32 was constructed previously.³² The target sequence of shRGC32 is 5′-GAT TCA CTT TAT AGG AAC AGC TT-3′. Ad-shRGC32 was generated as previously described.³² After incubation in a 37 °C 5% CO₂ incubator for 1 hour, the cells were gently washed with PBS for three times, and the number of adherent calcein AM-labeled (green fluorescent signal), or CM-DiI-labeled (red fluorescent signal) THP-1 cells were determined under a Nikon fluorescence microscope. Measurements were carried out for at least 10 different view fields.