Measuring oxygen levels in three different systems of Caco-2 cell culture.Caco-2 cells were cultured in three different systems, using conventional polystyrene 24-well plates, special 24-well gas permeable plates, or on membrane inserts in conventional plates. Optical sensor spots were used to measure dissolved O2 levels in these cultured cells over the course of 6 days under normoxia (143 mmHg) and for 6 hours under hypoxia (7 mmHg). Western blot analysis was used to determine the protein levels of hypoxia-inducible factor 1α (HIF-1α) in the different cultures.All culture systems displayed lower O2 levels over time than expected when cultured under normoxia conditions. On average, O2 levels reached as low as 25 mmHg in 24-well plates but remained at 97 and 117 mmHg in gas permeable plates and membrane inserts, respectively. Under hypoxia, 1 mL cell cultures equilibrated to 7 mmHg O2 within the first 60 minutes and dropped to 0.39 and 0.61 mmHg O2 in 24-well and gas permeable plates, respectively, after the 6-hour incubation period. Cultures in membrane inserts did not equilibrate to 7 mmHg by the end of the 6-hour incubation period, where the lowest O2 measurements reached 23.12 mmHg. Western blots of HIF-1α protein level in the whole cell lysates of the different Caco-2 cultures revealed distinct stabilization of HIF-1α after hypoxic incubation for 1, 2, and 4 hours in 24-well plates as well as gas permeable plates. For membrane inserts, notable HIF-1α was seen after 4 hours of hypoxic incubation.Cellular oxygen depletion was achieved in different hypoxic Caco-2 culture systems. However, different oxygen levels comparing different culture systems indicate that O2 level should be carefully considered in oxygen-dependent experiments.