Glucose colorimetric assay kit

The Extracellular Flux Analyzer XF96 (Seahorse Bioscience, Billerica, MA, USA) was used to measure cellular glycolysis capacity using Glycolysis Stress Test Kit (Seahorse Bioscience), according to the manufacturer’s instructions. Cells (4 × 10⁴ cells/well) were seeded in XF96 cell culture microplates. For ECAR assays, 10 mM glucose, 1 μM oligomycin and 100 mM 2-deoxy-glucose (2-DG) were added to evaluate ECAR values. The values of ECAR was normalized based on the numbers of cells and the curve was drawn. For glucose uptake and lactate production, cells were separately cultured in 6-well plate and incubated for 24 h, subsequently, cell supernatant was collected to calculate the glucose concentration and lactate level by using Glucose Colorimetric assay kit (BioVision, USA) and Lactate Colorimetric Assay Kit II (BioVision, USA), respectively. The data were normalized to total cell number of each sample.

LC cells with different GPC3 levels were seeded into 6-well plates at a density of 4 × 10⁵ cells/well. After incubation for 48 h, the culture medium was collected for glucose uptake with a glucose colorimetric assay kit (BioVision) following the manufacturer's protocol. The reduction of glucose concentration (glucose concentration at the beginning of cell culture minus the glucose concentration after 48 h of cell culture) in the culture media equates to the amount of glucose taken up by the liver cancer cells. For determination of the concentrations of lactate in the culture medium, a lactate assay kit (Sigma) was used and the detection was applied according to the manufacturer's protocol.
The final results of glucose uptake and lactate production were normalized on the basis of the total protein amounts of cells that were determined using a WonderOrange™ Protein Quantitation Kit (S-2014, US Everbright Inc).

Glucose, lactate, adenosine triphosphate (ATP) levels, and extracellular acidification rate (ECAR). The levels of glucose and lactate were calculated with a Glucose Colorimetric Assay Kit (BioVision, CA) and a Lactate Assay Kit (BioVision, CA) in line with the instructions of the manufacturer. ATP level was tested using Cell Titer-Glo Luminescent Cell Viability Assay (Promega, Madison, MI). ECAR was detected using Seahorse XF 96 Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA) according to the manufacturer’s instructions.

Following transfection, colon cancer cells (1 × 10⁶ cells/well) were incubated for 24 h. Glucose and lactate levels in the supernatants of cultured cells and its intracellular levels were measured using the Glucose Colorimetric Assay Kit (BioVision, Milpitas, USA) and Lactate Assay kit (BioVision, Milpitas, USA), respectively. Some cells were lyzed and the levels of G6PD enzymatic activity and intracellular NADPH in cell lysates were determined using G6PD Assay Kit (ab102529, Abcam, UK) and Amplite TM Colorimetric NADP/NADPH Ratio Assay Kit (ab65349, Abcam, UK), respectively.

Lactate secreted by cells was quantitated by Lactate Colorimetric Assay Kit (BioVision Inc Milpitas, CA). Lactate accumulation was calculated based on the formula: lactate level/number of cells. Glucose uptake by cells was quantitated by measuring the initial and final glucose content in the conditioned medium by Glucose Colorimetric Assay Kit (BioVision Inc). Glucose uptake rate was calculated based on the formula: ([initial glucose]-[final glucose])/time/number of cells. To confirm the glucose colorimetric assay, cells were stained with glucose analog 2-(N- (7-Nitrobenz-2-oxa- 1,3-diazol-4-yl) Amin)-2-Deoxyglucose (2-NBDG) (Life Technologies) followed by flow cytometry analysis. For ROS measurement, trypsinized cells were washed with PBS and stained with general ROS indicator chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA) (Life Technologies) and analyzed by flow cytometer. Data from flow cytometry studies were analyzed by FlowJo software. NADPH levels in cells were measured with NAPDH Quantification Colorimetric Kit (BioVision Inc).