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Measuring oxygen levels in Caco-2 cultures

Authors Zeitouni N, Fandrey J, Naim HY, von Köckritz-Blickwede M

Received 28 March 2015

Accepted for publication 9 July 2015

Published 9 October 2015 Volume 2015:3 Pages 53—66


Checked for plagiarism Yes

Review by Single-blind

Peer reviewer comments 5

Editor who approved publication: Prof. Dr. Dörthe Katschinski

Nathalie E Zeitouni,1 Joachim Fandrey,2 Hassan Y Naim,1 Maren von Köckritz-Blickwede1,3

1Department of Physiological Chemistry, University of Veterinary Medicine Hannover; 2Institute of Physiology, University Clinics Essen, University of Duisburg-Essen, Essen; 3Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine, Hannover, Germany

Purpose: 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 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.
Results: 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.
Conclusion: 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.

Keywords: hypoxia, normoxia, epithelial cell culture, hypoxia-inducible factor 1α

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