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