Proteomics characterisation of the L929 cell supernatant and its role in BMDM differentiation
Rachel E Heap1, José Luis Marín-Rubio1, Julien Peltier1
1Laboratory for Biological Mass Spectrometry, Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK.
Abstract:
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.
Insights
L929 cell-conditioned media (LCCM) contains more than just M-CSF, including immune-regulatory proteins. Macrophages differentiated with LCCM exhibit a stronger anti-inflammatory M1 phenotype compared to those differentiated with M-CSF alone.
Area of Science:
- Immunology
- Cell Biology
- Proteomics
Background:
- Bone marrow-derived macrophages (BMDMs) are crucial for in vitro macrophage research.
- Standard differentiation methods use M-CSF or L929 cell supernatant (LCCM).
- The composition of LCCM and its impact on BMDM phenotype remain unclear.
Purpose of the Study:
- To characterize the proteomic composition of LCCM over time.
- To investigate the effects of LCCM on BMDM differentiation and phenotype.
- To provide a resource for researchers using LCCM.
Main Methods:
- Quantitative mass spectrometry to analyze LCCM protein secretion kinetics over 2 weeks.
- Identification of 2,193 secreted proteins.
- Characterization of BMDM proteomes after differentiation with M-CSF, M-CSF + MIF, or LCCM.
Main Results:
- Identified 2,193 proteins in LCCM, including abundant M-CSF, MIF, osteopontin, Ccl2, and Ccl7.
- BMDMs differentiated with LCCM showed a more potent anti-inflammatory M1 phenotype than those differentiated with M-CSF.
- MIF, osteopontin, Ccl2, and Ccl7 were found at surprisingly high levels in LCCM.
Conclusions:
- LCCM is a complex mixture of immune-regulatory proteins, not just M-CSF.
- LCCM promotes a stronger M1 anti-inflammatory macrophage phenotype.
- This proteomic characterization of LCCM aids BMDM research.
