Analytical and Bioanalytical Chemistry

, Volume 411, Issue 4, pp 797–802 | Cite as

1H-NMR spectroscopy shows cellular uptake of HEPES buffer by human cell lines—an effect to be considered in cell culture experiments

  • Reinhard Depping
  • Karsten SeegerEmail author


HEPES is commonly used in cell culture media as a buffering substance. Compared to the bicarbonate/CO2 buffer system, it does not require a CO2 atmosphere, thereby ensuring stable pH values during handling of cell culture media outside of an incubator. Due to its intrinsic charge, HEPES is considered not to be taken up by cells, which was a prerequisite during buffer development for cell culture by Good and colleagues. However, during the last years, evidence has emerged that HEPES seems to be taken up into cells and that it has major effects on cellular functions. Investigating three different cell lines (MCF-7, U2OS, HeLa) showed that all of them accommodated HEPES-containing medium, i.e., they survive and proliferate in the presence of HEPES. Determination of intracellular metabolites revealed the presence of HEPES for all cell lines. Further analysis of MCF-7 cells showed that even 48 h after medium exchange from HEPES-containing medium to HEPES-free medium, intracellular HEPES could still be detected. Thus, contrary to the common view, HEPES is taken up by cells which should be taken into consideration for studies of specific cellular functions.

Graphical abstract


HEPES Cell culture NMR spectroscopy 



4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid


3-(Trimethylsilyl)propionic-2,2,3,3-d4 acid



The authors acknowledge Patricia Rouina for excellent technical support.

Compliance with ethical standards

Competing interests

The authors declare that they have no conflicts of interest.


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Institute of PhysiologyUniversity of LübeckLübeckGermany
  2. 2.Institute of Chemistry and MetabolomicsUniversity of LübeckLübeckGermany

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