Proteomics characterisation of the L929 cell supernatant and its role in BMDM differentiation

Abstract Bone marrow-derived macrophages (BMDMs) are a key model system to study macrophage biology in vitro. Commonly used methods to differentiate macrophages from bone marrow are treatment with either recombinant M-CSF or the supernatant of L929 cells, which secrete M-CSF. However, little is know...

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Published inbioRxiv
Main Authors Heap, Rachel E, Marín-Rubio, José Luis, Peltier, Julien, Heunis, Tiaan, Dannoura, Abeer, Moore, Adam, Trost, Matthias
Format Paper
LanguageEnglish
Published Cold Spring Harbor Cold Spring Harbor Laboratory Press 14.11.2020
Subjects
Bone marrow
CD11b antigen
CD11c antigen
Chemokines
Leukocyte migration
Macrophage colony-stimulating factor
Macrophage migration inhibitory factor
Mass spectroscopy
Migration inhibitory factor
Monocyte chemoattractant protein 1
Osteopontin
Phenotypes
Proteins
Proteomics
Regulatory proteins
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Summary:Abstract Bone marrow-derived macrophages (BMDMs) are a key model system to study macrophage biology in vitro. Commonly used methods to differentiate macrophages from bone marrow are treatment with either recombinant M-CSF or the supernatant of L929 cells, which secrete M-CSF. However, little is known about the composition of L929 cell conditioned media (LCCM) and how it affects BMDM phenotype. Here, we used quantitative mass spectrometry to characterise the kinetics of protein secretion from L929 cells over a two-week period, identifying 2,193 proteins. While M-CSF is very abundant in LCCM, we identified several other immune-regulatory proteins such as macrophage migration inhibitory factor (MIF), osteopontin and chemokines such as Ccl2 and Ccl7 at surprisingly high abundance levels. We therefore further characterised the proteomes of BMDMs after differentiation with M-CSF, M-CSF + MIF or LCCM, respectively. Interestingly, macrophages differentiated with LCCM induced a stronger anti-inflammatory M1 phenotype that those differentiated with M-CSF. This resource will be valuable to all researchers using LCCM for the differentiation of BMDMs. Competing Interest Statement The authors have declared no competing interest. Footnotes * small changes * Abbreviations BMDM bone marrow-derived macrophages FBS foetal bovine serum GO Gene Ontology iBAQ Intensity Based Absolute Quantification LCCM L929 Cell Conditioned Media LC-MS liquid chromatography mass spectrometry M-CSF macrophage colony-stimulating factor-1 MIF macrophage migration inhibitory factor TEAB triethyl ammonium bicarbonate TFA Trifluoro acetic acid TMT tandem mass tag
DOI:10.1101/2020.08.20.259515