(A) Effects of three different oxygen tensions (20%, 2.0% and 0.5%) on the cellular metabolic activity of human oral keratinocytes up to 96 hours. The metabolic activity was assessed using a Cell-Counting kit-8 (N=10). Assays were performed in triplicate. Asterisks represent statistically significant differences determined by Steel-Dwass test (*p< 0.05).
(B) Effects of three different oxygen tensions on the proliferation rate of human oral keratinocytes up to 6 days. Viable cells were stained with trypan blue and counted (N=8). Assays were performed in duplicate. Asterisks represent statistically significant differences determined by Tukey’s post-hoc test (*p< 0.05, **p< 0.01).
(C) Effects of three different oxygen tensions on the colony forming efficiency of human oral keratinocytes. Cells were stained with crystal violet and the number of colonies were microscopically and macroscopically counted (N=9) as stated in the Materials and Methods. An asterisk represents statistically significant difference determined by Tukey’s post-hoc test (*p< 0.05). The statistical differences between normoxic and hypoxic cells were marginal except the microscopic quantification between 2.0% and 20%O2 culture condition.
(D) Representative images of clonogenic assay. After cells were cultured for 4 days at 20, 2.0 and 0.5% O2, colonies were fixed and stained with Crystal Violet as aforementioned.
(E) CFSE dye profile of cells cultured in normoxic and hypoxic conditions for 48 h. Their patterns were diverse among different oxygen tensions as well as individuals. The data shown are representative of six separate experiments. (a) 20% O2, (b) 2% O2, (c) 0.5% O2, (d) Overlay, (e) FITC standard beads. The values shown in (a), (b), (c) are the percentage of cells that could be experienced round of divisions of 2, 1 and 0 times according to the protocol of Chadli’s work.