title: Measuring oxygen levels in Caco-2 cultures.
abstract: OBJECTIVE
  Measuring oxygen levels in three different systems of Caco-2 cell culture.
  
  
  METHODS
  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 O2levels 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.
  
  
  RESULTS
  All culture systems displayed lower O2levels over time than expected when cultured under normoxia conditions. On average, O2levels 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 O2within the first 60 minutes and dropped to 0.39 and 0.61 mmHg O2in 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 O2measurements 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.
  
  
  CONCLUSIONS
  Cellular oxygen depletion was achieved in different hypoxic Caco-2 culture systems. However, different oxygen levels comparing different culture systems indicate that O2level should be carefully considered in oxygen-dependent experiments.
authors: Department of Physiological Chemistry, University of Veterinary Medicine Hannover.
authors: Nathalie E
authors: NE
authors: Zeitouni
authors: Institute of Physiology, University Clinics Essen, University of Duisburg-Essen, Essen.
authors: Joachim
authors: J
authors: Fandrey
authors: Department of Physiological Chemistry, University of Veterinary Medicine Hannover.
authors: Hassan Y
authors: HY
authors: Naim
authors: Department of Physiological Chemistry, University of Veterinary Medicine Hannover; Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine, Hannover, Germany.
authors: Maren
authors: M
authors: von Köckritz-Blickwede
keywords: epithelial cell culture; hypoxia; hypoxia-inducible factor 1α; normoxia