RT Journal Article SR Electronic T1 Proteomics characterisation of the L929 cell supernatant and its role in BMDM differentiation JF Life Science Alliance JO Life Sci. Alliance FD Life Science Alliance LLC SP e202000957 DO 10.26508/lsa.202000957 VO 4 IS 6 A1 Rachel E Heap A1 José Luis Marín-Rubio A1 Julien Peltier A1 Tiaan Heunis A1 Abeer Dannoura A1 Adam Moore A1 Matthias Trost YR 2021 UL https://www.life-science-alliance.org/content/4/6/e202000957.abstract AB BMDMs are a key model system to study macrophage biology in vitro. Commonly used methods to differentiate macrophages from BM 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 the BMDM phenotype. Here, we used quantitative mass spectrometry to characterise the kinetics of protein secretion from L929 cells over a 2-wk period, identifying 2,193 proteins. Whereas 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.