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

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. While MIF has no significant effect on the BMDM proteome, LCCM induced a slightly pre-activated phenotype and the expression of a number of known innate immune proteins in macrophages. Interestingly, LCCM induced higher expression of CD11b, while BMDMs differentiated with M-CSF alone showed higher expression of CD11c. This resource will be valuable to all researchers using LCCM for the differentiation of BMDMs.