C 12271

HAoECs were purchased from PromoCell (C-12271). Cells were cultured at 37°C and 5% CO₂ in endothelial cell growth medium MV2 (C-22020, PromoCell) and passaged after reaching confluence. Cells from passages 3 to 6 were used in the study.

THP-1 were transfected with GPR26 GapmeRs for 24, 48 and 72 h. At the end of the experiment, cells were labeled with 5 μM/L × 10⁶ cells calcein-AM (Calcein, AM, Zellfarbstoff, C1430, Invitrogen, Life Technologies) for 45 min as previously described [31 (link)], with some modifications. Human Aortic Endothelial cells (HAoECs criopreserved, 500.000 cells, C-12271, PromoCell, Heidelberg, Germany) were cultured on gelatin-coated 96-well black plates. Confluent HAoEC monolayers were co-cultured with fluorescently labeled THP-1 cells for 20 min at 37 °C. Then, the monolayers were washed three times with PBS to remove non-adherent cells. Fluorescence was acquired at 492 nm (excitation) and 535 nm (emission) on a plate reader (Tecan). Relative fluorescence from treated monocytes adherent to HAoECs was normalized on that from the relative monocytes control groups. Comparisons were made between all four groups, as indicated in the Results. Representative images were acquired with a Thunder Imager Live Cell inverted microscope (Leica microsystem, Leica Mikrosysteme Vertrieb GmbH, Wetzlar, Germany) and equipped with a dual fluorescent and brightfield camera. Images were acquired for each of the 4 technical replicates and for all 3 biological replicates.