TY - JOUR
T1 - Reduced quenching and extraction time for mammalian cells using filtration and syringe extraction
AU - Hernández Bort, Juan A.
AU - Shanmukam, Vinoth
AU - Pabst, Martin
AU - Windwarder, Markus
AU - Neumann, Laura
AU - Alchalabi, Ali
AU - Krebiehl, Guido
AU - Koellensperger, Gunda
AU - Hann, Stephan
AU - More Authors, null
PY - 2014
Y1 - 2014
N2 - In order to preserve the in vivo metabolite levels of cells, a quenching protocol must be quickly executed to avoid degradation of labile metabolites either chemically or biologically. In the case of mammalian cell cultures cultivated in complex media, a wash step previous to quenching is necessary to avoid contamination of the cell pellet with extracellular metabolites, which could distort the real intracellular concentration of metabolites. This is typically achieved either by one or multiple centrifugation/wash steps which delay the time until quenching (even harsh centrifugation requires several minutes for processing until the cells are quenched) or filtration. In this article, we describe and evaluate a two-step optimized protocol based on fast filtration by use of a vacuum pump for quenching and subsequent extraction of intracellular metabolites from CHO (Chinese hamster ovary) suspension cells, which uses commercially available components. The method allows transfer of washed cells into liquid nitrogen within 10-15. s of sampling and recovers the entire extraction solution volume. It also has the advantage to remove residual filter filaments in the final sample, thus preventing damage to separation columns during subsequent MS analysis. Relative to other methods currently used in the literature, the resulting energy charge of intracellular adenosine nucleotides was increased to 0.94 compared to 0.90 with cold PBS quenching or 0.82 with cold methanol/AMBIC quenching.
AB - In order to preserve the in vivo metabolite levels of cells, a quenching protocol must be quickly executed to avoid degradation of labile metabolites either chemically or biologically. In the case of mammalian cell cultures cultivated in complex media, a wash step previous to quenching is necessary to avoid contamination of the cell pellet with extracellular metabolites, which could distort the real intracellular concentration of metabolites. This is typically achieved either by one or multiple centrifugation/wash steps which delay the time until quenching (even harsh centrifugation requires several minutes for processing until the cells are quenched) or filtration. In this article, we describe and evaluate a two-step optimized protocol based on fast filtration by use of a vacuum pump for quenching and subsequent extraction of intracellular metabolites from CHO (Chinese hamster ovary) suspension cells, which uses commercially available components. The method allows transfer of washed cells into liquid nitrogen within 10-15. s of sampling and recovers the entire extraction solution volume. It also has the advantage to remove residual filter filaments in the final sample, thus preventing damage to separation columns during subsequent MS analysis. Relative to other methods currently used in the literature, the resulting energy charge of intracellular adenosine nucleotides was increased to 0.94 compared to 0.90 with cold PBS quenching or 0.82 with cold methanol/AMBIC quenching.
KW - CHO cells
KW - Fast filtration
KW - Metabolite extraction
KW - Metabolomics
KW - Quenching
UR - http://www.scopus.com/inward/record.url?scp=84902121319&partnerID=8YFLogxK
U2 - 10.1016/j.jbiotec.2014.04.014
DO - 10.1016/j.jbiotec.2014.04.014
M3 - Article
C2 - 24794799
AN - SCOPUS:84902121319
SN - 0168-1656
VL - 182-183
SP - 97
EP - 103
JO - Journal of Biotechnology
JF - Journal of Biotechnology
IS - 1
ER -