Dataset Information

Addressing reproducibility challenges in cell culture: the impact of oxylipins contained in fetal calf serum

ABSTRACT: Reproducibility issues regarding complex in vitro cell culture experiments have been mainly attributed to genetic variations within cell lines. Batch-dependent fetal calf serum variations are well known and also represent relevant influencing factors. However, the molecular constituents mediating such effects remained largely unknown. High resolution mass spectrometry is a powerful tool to identify molecular FCS constituents and investigate their effect on cultured cells. Using a differentiation and inflammatory stimulation protocol on U937 cells we observed FCS batch-dependent variations of secreted cytokines, oxylipins and other inflammatory mediators. In order to detect candidate bioactive molecules contained in FCS, the protein and oxylipin composition of FCS batches was investigated. Remarkably, the protein composition showed some, but much less batch-dependent variation when compared to the oxylipin composition. Efficient uptake of C13-labelled arachidonic acid from medium by U937 macrophages and inflammation-induced release thereof including oxylipin products was evidenced. Balancing out FCS batch-dependent nanomolar concentration differences of two selected oxylipins, 5-HETE and 15-HETE, by spiking experiments, resulted in significant proteome alterations of cultured U937 macrophages indicating PPAR activation. High-resolution microscopy demonstrated HETE-induced formation of peroxisomes, thus independently corroborating the proteome profiling results. In conclusion, the present data demonstrate a strong and previously underappreciated influence of FCS-contained oxylipins on cellular effector functions, thus representing a plausible cause for disturbing reproducibility issues.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Macrophage

DISEASE(S): Acute Leukemia

SUBMITTER: Christopher Gerner

LAB HEAD: Christopher Gerner

PROVIDER: PXD020617 | Pride | 2021-06-01

REPOSITORIES: Pride

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Publications

Eicosanoid Content in Fetal Calf Serum Accounts for Reproducibility Challenges in Cell Culture.

Niederstaetter Laura L Neuditschko Benjamin B Brunmair Julia J Janker Lukas L Bileck Andrea A Del Favero Giorgia G Gerner Christopher C

Biomolecules 20210115 1

Reproducibility issues regarding in vitro cell culture experiments are related to genetic fluctuations and batch-wise variations of biological materials such as fetal calf serum (FCS). Genome sequencing may control the former, while the latter may remain unrecognized. Using a U937 macrophage model for cell differentiation and inflammation, we investigated whether the formation of effector molecules was dependent on the FCS batch used for cultivation. High resolution mass spectrometry (HRMS) was ...[more]

PMID: 33467719

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