SILAC labeling of mouse bone marrow-derived macrophage and mass spectrometry

Aim Mouse bone marrow-derived macrophages(BMM) are recognized as standard model cells of macrophages,thus serving as a key tool and objective cell type in pharmacology.Due to limited proliferation capacity of macrophages,metabolically labeling BMM remains as a challenge in macrophage biological investigations.Therefore,the focus of this study was to label BMM with stable isotope labeling with amino acids in cell culture(SILAC).Methods Methods include:mouse bone marrow isolation,6-day differentiation with M-CSF to prepare BMM;meanwhile,to use SILAC to label global proteins,followed by cell lysis,SDS-PAGE separation,in-gel digestion,mass spectrometry and bioinformatics.Results On Day 6 post-differentiation,96.5% of total mouse bone marrow cells were observed to become mature BMM.70 ku band in SDS-PAGE separation was adopted to test the labeling efficiencies.The number of heavy lysine labeled proteins were 18,12 and 13 for the time points of Day 6,8 10 post-differentiation.Eight proteins were repeatedly identified in all time points.Statistically,the labeling efficiencies of the three time points were(90.62±0.03)%,(90.23±0.03)% and(90.40±0.02)%,respectively.Conclusion These results ensure the feasibility of employing SILAC-based proteomics in macrophage-relevant pharmacological investigations.