pCMV-SPORT6 transient transfection

1.5 × 10⁶ Raw-264.7 cells were seeded in a 6-well plate in complete DMEM and left overnight to propagate. The following day, fresh serum-free DMEM (without antibiotic) was added to the cells for transfection. Overexpression plasmids were purchased as pCMV-SPORT-6-Arg2 and pCMV-SPORT6-Arg1 (Invitrogen). They were transfected at 500 ng/well in the respective wells using Lipofectamine 3000 (Invitrogen). For control wells, an empty vector pCMV-SPORT6 plasmid was used. Cells were incubated at 37 °C and 5% CO₂ for 4 h with the transfection mix in serum and antibiotic free media, after which media was replaced with complete DMEM. Cells were incubated for a further 24 h for Immunoblotting. For metabolic profiling assays, cells were scraped 24 hours post-transfection, counted for number and viability and transferred to XF_e96-well plates (Agilent) for another 24 h before conducting the assay.