Hexokinase colorimetric assay kit

Hexokinase Colorimetric Assay Kit, Glucose Uptake Colorimetric Assay kit, ATP Colorimetric Assay kit and Lactate Assay Kit II were purchased from Biovision and used to detect HK activity, glucose uptake, ATP, and lactate production, respectively. These assays were detected following the manufacturer’s protocols as described previously [47 (link)].

The enzyme activities of HK, PFK, and PK in cancer cells were measured using a hexokinase colorimetric assay kit (BioVision), phosphofructokinase (PFK) activity colorimetric assay kit (BioVision), and pyruvate kinase activity colorimetric/fluorometric assay kit (BioVision), respectively, following the manufacturer’s protocols.

For the HK activity assay, cells (1 × 10⁵) were harvested and homogenized with 200 μL ice-cold HK Assay Buffer (BioVision) for 10 minutes. The cells were centrifuged at 15,000g for 5 minutes, and the supernatant was assayed using a Hexokinase Colorimetric Assay Kit (BioVision). The reaction mixture was incubated at room temperature for 20–60 minutes and measured at 450 nm in a microplate reader (TECAN).
For the PFK activity assay, cells (1 × 10⁵) were harvested and homogenized with 200 μL ice-cold PFK Assay Buffer (MilliporeSigma) for 10 minutes. The cells were centrifuged at 13,000g for 10 minutes, and the supernatant was assayed by a Phosphofructokinase Activity Colorimetric Assay kit (MilliporeSigma). The reaction mixture was incubated at room temperature and measured at 450 nm every 5 minutes.
For the PK activity assay, cells (1 × 10⁵) were harvested and extracted with 4 volumes of the assay buffer (BioVision). The cells were centrifuged at 15,000g for 10 minutes to obtain a clear extract. After centrifugation, the supernatant was assayed by a Pyruvate Kinase Activity Assay Kit (BioVision). The reaction mixture was incubated for 10–20 minutes at room temperature and measured at 570 nm with a microplate reader.