Tracing Metabolic Dynamics in Cell Activation

For tracing experiments, MDSC + ISO and MDSC + PBS in one set and β2-AR^-/- + ISO and β2-AR^-/- + PBS in another set were generated using rmIL-6 and rmGM-CSF (40 ng/mL each) for 4 days. On day 4, the cells were incubated with tracing media: Gln-free RPMI medium supplemented with 10% dialyzed FBS (Life Technologies), 20 mM HEPES, 0.05 mM 2-mercaptoethanol, and 1% penicillin-streptomycin plus 2 mM ¹³C₅-glutamine (¹³C₅-Gln) (Cambridge Isotope Laboratories,). At 2 h, 8 h. and 24 h. incubation timepoints, the cells were washed twice with PBS, once quickly with distilled water, and quenched with 60% cold acetonitrile in water. Polar metabolites were extracted by the solvent partitioning method with a final CH₃CN:H₂O:CHCl₃ (2:1.5:1, v/v) ratio, followed by a second extraction including methanol, and lyophilized as previously described^{73 (link),74} .