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

Abstract

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