Culturing and Proliferation of Human Cell Lines

HDFs (PromoCell GmbH, Heidelberg, Germany) and HeLa cells (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and 0.1% antibiotics (Gibco) at 37℃ in a 5% CO₂ incubator (APM-30D; ASTEC, Fukuoka, Japan) [33 (link)]. Oxygen levels of 21%, as the normoxic condition, and 1%, as the hypoxic condition, were used for culturing HDFs from passage 4 to passage 6 [33 (link)]. When the cell confluency of all cells reached 90%, the cells were passaged using 0.25% Trypsin-EDTA (Gibco).
For the proliferation assay, 2 × 10⁵ HDFs at passage 6 were cultured in a 100-mm culture plate for 5 days, and cell numbers were measured using trypan blue 0.5% solution staining (Biowest, Riverside, MO, USA). Other cervical cancer cells (CaSki, ME-180, and SiHa cells), melanoma cells (A375SM), breast cancer cells (MCF-7), and lung cancer cells (NCI-H358) were purchased from the Korea Cell Line Bank (Seoul, Korea). CaSki, ME-180, A375SM, MCF-7, and NCI-H358 were cultured in RPMI1640 (Gibco) supplemented with 10% FBS (Gibco), 1% L-glutamine (Gibco), 25 mM HEPES (Gibco), 25 mM NaHCO₃ (Gibco), and 0.1% antibiotics (Gibco). SiHa cells were cultured in DMEM supplemented with 10% FBS (Gibco) and 0.1% antibiotics (Gibco).