SILAC Labeling and Macrophage Differentiation

THP-1 monocytes were purchased from the German collection of microorganisms and cell cultures (DSMZ). Stable isotope labeling with amino acids in cell culture (SILAC, Ong et al., 2002 (link), 2003 (link)) was achieved by propagating cells in RPMI 1640 free of L-lysine, L-arginine and L-glutamine (PAA Laboratories) supplemented with 220 μM L-lysine and 144 μM L-arginine in either their light (Lys-0 and Arg-0) or heavy isotope-labeled forms (¹³ ${\text{C}}_6^{15}$ N₂-L-lysine/Lys-8 and ¹³ ${\text{C}}_6^{15}$ N₄-L-arginine/Arg-10) (Silantes), 2 mM L-glutamine (Sigma-Aldrich), 10% heat-inactivated dialyzed fetal calf serum (FCS) (Sigma-Aldrich) and the antibiotics penicillin and streptomycin (Biochrom) at concentrations of 100 units/ml and 100 μg/ml respectively. Cell culturing was performed at 37°C and 5% CO₂ in a humidified atmosphere. Cells were grown for at least six cell doublings to ensure complete incorporation of labeled amino acids. For differentiation, phorbol-12-myristate 13-acetate (PMA) (Sigma-Aldrich) was added to a final concentration of 100 nM. After 3 days, the PMA supplemented media was removed, cells were washed with PBS and rested in fresh PMA-free media for further 24 h in order to obtain phenotypic characteristics of macrophages (Daigneault et al., 2010 (link)).