Phosphofructokinase-1 Assay

Cell lysate was obtained using the manufacturer protocol of the PFK1 assay kit (abcam). Briefly, cells were washed with cold PBS. They were then resuspended in 100μL of cold assay buffer. Cells were homogenized using vigorous pipetting, and then incubated on ice for 10 minutes. The samples were then centrifuged at 12000g for 5 minutes at 4°C. The supernatant was then collected and then used in the PFK1 activity assay (abcam).

Briefly, the cell lysate was incubated in a reaction well for a one-hour time period. The absorbance at a certain time point reflected the concentration of NADH in the reaction well. NADH in the reaction well is produced through a reaction from the assay kit’s enzyme mix, substrate, and ADP produced by PFK1 in the cell lysate. The PFK1 in the cell lysate converts fructose 6-phosphate and ATP into fructose 1,6-diphosphate and ADP; the ADP from PFK1 reactions combines with NAD⁺ to form AMP and NADH. The concentration of NADH was used later for calculating the enzymatic activity.

Two time points that recorded maximum changes in NADH concentration during the assay were selected. The V_max was calculated using the change in NADH concentration during these time points. One unit of PFK1 activity is equivalent to the amount of PFK1 that will generate 1 μmol of NADH per minute. Triplicate samples were used from each culture for cytokine and enzyme activity assays.