2.5. Colony Forming Unit (CFU) Assay

Cells grown at 3% or 21% O₂ were used at early passage (P2) and late passage (between P11 to P22). They were seeded at a clonal density of 5 cells/cm² of a six-well plate in α-MEM supplemented with 10% fetal bovine serum (FBS) and incubated under a humidified atmosphere of 5% CO₂ at 21% O₂. After seven days, cells were fed with fresh medium for an additional period of seven days. At day 14, colony formation was obvious and the assay was stopped by fixation with cold absolute methanol for 10 min at 4 °C. Colonies were stained for 10 min in 0.5% crystal violet prepared in 25% methanol. After washing, colonies with more than 50 cells were scored by the use of a Leica MZ10F magnifying device. Results were analyzed by calculation of cloning efficiency E (E (%) = (Number of colonies/number of seeded cells) × 100).