4.8. Pyruvate Kinase Activity Assay

The pyruvate kinase (PK) activity assay kit (Abcam, Cambridge, UK, Ab83432) was used to measure pyruvate kinase activity between the control and the sepsis groups. All procedures were followed according to the manufacturer’s manual. In brief, 1 × 10⁶ cell pellets were prepared to extract with four volumes of assay buffer and bring the volume to 50 μL/well with PK assay buffer of each sample. The standard linear curve was generated by a serial dilution of 1 nmol/μL of a standard pyruvate solution. It was calculated using the amount of enzyme that transferred a phosphate group from phosphoenolpyruvate (PEP) to ADP, yielding 1.0 μmol of pyruvate per minute at 25 °C. All reacted standards and samples were subjected to colorimetric measurement at 570 nm of a microplate reader.