Influence of washing and quenching in profiling the metabolome of adherent mammalian cells: A case study with the metastatic breast cancer cell line MDA-MB-231
Metabolome characterisation is a powerful tool in oncology. To obtain a valid description of the intracellular
metabolome, two of the preparatory steps are crucial, namely washing and quenching. Washing
must effectively remove the extracellular media components and quenching should stop the metabolic
activities within the cell, without altering the membrane integrity of the cell. Therefore, it is important to
evaluate the efficiency of the washing and quenching solvents. In this study, we employed two previously
optimised protocols for simultaneous quenching and extraction, and investigated the effects of a number
of washing steps/solvents and quenching solvent additives, on metabolite leakage from the adherent
metastatic breast cancer cell line MDA-MB-231. We explored five washing protocols and five quenching
protocols (including a control for each), and assessed for effectiveness by detecting ATP in the medium
and cell morphology changes through scanning electron microscopy (SEM) analyses. Furthermore, we
studied the overall recovery of eleven different metabolite classes using the GC-MS technique and compared
the results with those obtained from the ATP assay and SEM analysis. Our data demonstrate that a
single washing step with PBS and quenching with 60% methanol supplemented with 70 mM HEPES
(−50 °C) results in minimum leakage of intracellular metabolites. Little or no interference of PBS (used in
washing) and methanol/HEPES (used in quenching) on the subsequent GC-MS analysis step was noted.
Together, these findings provide for the first time a systematic study into the washing and quenching
steps of the metabolomics workflow for studying adherent mammalian cells, which we believe will
improve reliability in the application of metabolomics technology to study adherent mammalian cell
metabolism