Phosphofructokinase activity colorimetric assay kit

PFK activity was assessed using Phosphofructokinase Activity Colorimetric Assay Kit (MAK093, Sigma) according to the manufacturer’s instruction. In all, 2 × 10⁶ of indicated cells were used for this experiment and the PFK activity is calculated by the absorbance of 450 nm using Multiscan GO spectrophotometer (Thermo Fisher Scientific, Vantaa, Finland).

Phosphofructokinase measurements were done according to the manufacturer’s datasheet (Phosphofructokinase Activity Colorimetric Assay Kit, Sigma, Switzerland). SH-SY5Y cells were seeded in T25 flasks (Techno Plastic Products AG (TPP), Trasadingen, Switzerland) at a density of 3 × 10⁶ cells per flask and treated as previously described above. Briefly, the protein was extracted by scraping off cells in 1 mL HBSS and centrifuged at 13.200×g for 10 min. The buffer was removed and the pellet was dissolved in 200 µL cold PFK assay buffer and centrifuged at 13.200×g for 10 min. The pellet was solubilized in 60 µL cold PFK buffer and 50 µL were transferred to a 96- well plate and a master mix consisting of PFK Assay Buffer, PFK Enzyme Mix, PFK Developer, ATP and PFK substrate was added. The absorbance (450 nm) was measured using a Synergy H1 multi-mode reader. The data were compared with the NADH standard curve ranging from [2 to 10 nmol]/well and values were normalized to 1 mg protein. The protein amount of each sample was determined with Pierce™ 660 nm Protein Assay Reagent (Thermo Fisher Scientific, Switzerland).