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Reduced quenching and extraction time for mammalian cells using filtration and syringe extraction.

ABSTRACT: 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 10-15s 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.

SUBMITTER: Hernandez Bort JA

PROVIDER: S-EPMC4071440 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Reduced quenching and extraction time for mammalian cells using filtration and syringe extraction.

Hernández Bort Juan A JA Shanmukam Vinoth V Pabst Martin M Windwarder Markus M Neumann Laura L Alchalabi Ali A Krebiehl Guido G Koellensperger Gunda G Hann Stephan S Sonntag Denise D Altmann Friedrich F Heel Christine C Borth Nicole N

Journal of biotechnology 20140429

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 ...[more]

PMID: 24794799

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Project description:Over 20% of the world's tropical forests have been selectively logged, and large expanses are allocated for future timber extraction. Reduced-impact logging (RIL) is being promoted as best practice forestry that increases sustainability and lowers CO2 emissions from logging, by reducing collateral damage associated with timber extraction. RIL is also expected to minimize the impacts of selective logging on biodiversity, although this is yet to be thoroughly tested.We undertake the most comprehensive study to date to investigate the biodiversity impacts of RIL across multiple taxonomic groups. We quantified birds, bats and large mammal assemblage structures, using a before-after control-impact (BACI) design across 20 sample sites over a 5-year period. Faunal surveys utilized point counts, mist nets and line transects and yielded >250 species. We examined assemblage responses to logging, as well as partitions of feeding guild and strata (understorey vs. canopy), and then tested for relationships with logging intensity to assess the primary determinants of community composition.Community analysis revealed little effect of RIL on overall assemblages, as structure and composition were similar before and after logging, and between logging and control sites. Variation in bird assemblages was explained by natural rates of change over time, and not logging intensity. However, when partitioned by feeding guild and strata, the frugivorous and canopy bird ensembles changed as a result of RIL, although the latter was also associated with change over time. Bats exhibited variable changes post-logging that were not related to logging, whereas large mammals showed no change at all.Indicator species analysis and correlations with logging intensities revealed that some species exhibited idiosyncratic responses to RIL, whilst abundance change of most others was associated with time.Synthesis and applications. Our study demonstrates the relatively benign effect of reduced-impact logging (RIL) on birds, bats and large mammals in a neotropical forest context, and therefore, we propose that forest managers should improve timber extraction techniques more widely. If RIL is extensively adopted, forestry concessions could represent sizeable and important additions to the global conservation estate - over 4 million km2.

Dataset Information

Reduced quenching and extraction time for mammalian cells using filtration and syringe extraction.

Publications

Reduced quenching and extraction time for mammalian cells using filtration and syringe extraction.

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