Cell Culture Protocol for HEK293 and HUVEC

HEK293 were grown in DMEM containing glucose 1 g/l, pyruvate and GlutaMAX and supplemented with 10% fetal bovine serum, 1% non-essential amino acids, HEPES 10 mM and 1% penicillin and streptomycin (all from Life Technologies) at 37°C in a water humidified 5% CO₂ incubator. Cells were sub-cultured twice a week at 90% confluence. HUVEC (passages 1 to 5) were obtained from Gibco and cultivated as described in Ruiz et al. (2017) (link). Briefly, cells were grown in M200 medium (Gibco) containing the Low Serum Growth Supplement (Gibco) and 1% penicillin and streptomycin. Cells were sub-cultured twice a week at 90% confluence, and cultivated on treated plastic flask and multi-dish plates (Nunc). For FRAP and Laurdan dye experiments, HEK293 or HUVEC were seeded in glass bottom dishes (Ibidi) pre-coated with 0.1% porcine gelatin (Sigma). The HEK293 cell line was authenticated by genotyping and confirmed to be free of mycoplasma.