Lncap prostate cancer cell line

HeLa cervical cancer cell line and LNCaP prostate cancer cell line were obtained from ATCC (Rockville, MD, USA). UT-SCC74 primary cell line derived from human head and neck squamous cell carcinoma (HNSCC) was kindly provided by Prof Reidar Grénman. HeLa cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Sigma-Aldrich), UT-SCC74 cells in Minimun Essential Medium Eagle with alpha modification (α-MEM, Gibco) and LNCaP cells in Roswell Park Memorial Institute medium (RPMI-1640, Gibco). All media were supplied with 10% fetal calf serum (FCS), L-glutamine and antibiotics (penicillin and streptomycin). Cells were cultured in 37 °C in air/5% CO₂ and tested negative for mycoplasma once a month. The reference cells for the Minihypoxy system were cultured in 1% oxygen and 5% CO₂ in the hypoxia workstation (Invivo2 400, Ruskinn Technology). The reference cells were plated on Minihypoxy culture chambers attached to glass substrate. Chambers were covered with the glass lid and kept on Petri dishes during incubation and for convenience in handling. For live cell imaging the cells were transfected with EGFP expression vector. Transfection reagent Lipofectamine 3000 (Invitrogen) was used according to manufacturer’s specifications. The images were obtained with LSM780 microscope (Carl Zeiss, Oberkochen, Germany).