Figure 1. Sustained and cumulative expression of VEGF in hypoxia and atmospheric oxygen. Detection of VEGF levels in hypoxia with ELISA. HLE-B3 cells were cultured in 25 cm² flasks with 20% FBS and switched to serum-free media 24 h before the experiment. The cells were incubated with 3 ml of serum-free media in hypoxia (1% oxygen) or remained in atmospheric oxygen (about 21% oxygen) for up to 72 h. Cell-free supernatants were collected in triplicate at 8, 24, 48, and 72 h and analyzed with ELISA to detect the VEGF levels. VEGF consistently accumulated throughout the 72 h incubation period regardless of whether the cells were maintained in hypoxia or atmospheric oxygen (p<0.05). A Student t test was performed to compare the VEGF levels between hypoxia and normoxia. Significantly higher levels of VEGF were detected at all-time points beyond the initial 8h point in hypoxia compared with atmospheric oxygen. Error bars are not shown at time points because the symbol is larger than the error bar.