Lipofectamine® 2000 Transfection Reagent

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	Do not add antibiotics to the Transfection Medium

Protocol tips
Do not add antibiotics to the Transfection Medium

Publication protocol

Transient Transfection and 3‐(4,5‐Dimethylthiazol‐2‐yl) 2,5‐Diphenyl Tetrazolium Bromide Assay
3T3‐L1 preadipocytes (1 × 10/well) were seeded and maintained overnight in 10% BCS DMEM‐F12 medium. Lipofectamine 2000 (Invitrogen Life Technologies, Gran Island, NY, USA) was used to carry out the pcDNA3.1(–)‐mZAG expression plasmid transfection experiment. The DNA concentration in the transfection reagent was 0.5 μg/μL. The cell growth was monitored at 24, 36, 48, 72, 96, 120 and 144 h after the transfection using the 3‐(4,5‐Dimethylthiazol‐2‐yl) 2,5‐diphenyl tetrazolium bromide (MTT) method, as described previously. For the 96, 120 and l44 h experimental time‐points, a second transfection was carried out 72 h after the first transfection. Briefly, the cells were washed, 50 μL MTT (3‐[4,5‐dimethylthiazol‐2‐yl] 2,5‐diphenyl tetrazolium bromide; 1 mg/mL) was added, and the cells were incubated at 37°C for 4 h. A cell lysis solution (100 μL/well, including 10% w/v sodium dodecyl sulfate [SDS], 5% isobutanol and 0.01 N HCL) was added, and the cells were incubated overnight. The optical density (OD) at 620 nm was recorded using an enzyme‐linked immunosorbent assay (ELISA) reader (Anthos Labtec, Wals, Austria).

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