2.2. Cell culture and transfection

We used an immortalized human umbilical vein endothelial cell (HUVEC) line. In the published Methods, these cells were maintained in RPMI medium supplemented with 10% fetal bovine serum (FBS), streptomycin, and penicillin, at 37 °C in 5% CO₂.

For the migration and adhesion experiments in the paper, cells were first grown in complete RPMI and then switched to RPMI containing 0.2% BSA during the assay phase. For migration assays, HUVECs were seeded at 5 × 10^5 cells/well in 6-well plates. After 2 days, the medium was replaced with RPMI + 0.2% BSA, and 24 h later a scratch was made. The cells were then incubated for another 24 h with the indicated treatments. For adhesion assays, cells were harvested and seeded at 2.5 × 10^5 cells/well in 96-well plates and incubated for 24 h in RPMI + 0.2% BSA.

Regarding routine maintenance after thawing, this part was not described in detail in the paper, but in our lab we usually handle the immortalized HUVECs as follows: our cryovials contain 2 × 10^6 cells frozen in 10% DMSO, 50% RPMI, and 40% FBS using Mr. Frosty freezing containers at -80 °C. The cells are thawed directly into RPMI medium, centrifuged at 1400 rpm for 10 min at room temperature to remove DMSO, resuspended in fresh complete RPMI medium, and plated in a T75 flask. The medium is replaced 2 days after thawing, and the cells are then monitored and expanded as needed. They are typically ready for experiments the following week.

In our hands, we usually use these cells for up to 10 passages after thawing.