Glutathione assay kit

To measure total GSH levels, LX-2 cells was seeded onto 6-well plates with a density of 1 × 10⁵ cells per well and treated by DOX (10, 25 μM) for 48 h. The samples were determined using a glutathione assay kit (Nanjing Jiancheng Bioengineering Institute, China) following a standard protocol.

Cells were cultured in 35-mm culture dishes for 6 h or overnight, then the culture medium was replaced by fresh complete medium, and the cells were incubated for an additional 48 h. The media were collected for measurements of the glucose and lactate concentrations, and the cells were harvested to obtain protein lysates and measure GSH and ATP concentrations. The glucose levels were determined using a glucose assay kit (Nanjing Jiancheng), and glucose consumption was calculated by deducting the measured glucose concentration in the media from the original glucose concentration. The lactate levels were determined using a lactate assay kit (Nanjing Jiancheng). The intracellular GSH and ATP levels were measured using a glutathione assay kit (Nanjing Jiancheng) and an ATP concentration assay kit (Nanjing Jiancheng), respectively. All the values were normalized based on the total protein concentration obtained using the Bradford protein assay.
The levels of ECAR and OCR were assessed using an XFe96 Analyzer (Seahorse Bioscience). Cells were cultured in a Seahorse XF cell culture microplate at a density of 2 × 10⁴ cells/well (U2R) or 1 × 10⁴ cells/well (U2OS).

The glutathione (GSH) concentration was determined using a glutathione assay kit purchased from Jiancheng Bioengineering institute and carried out according to the manufacturer s instructions. The treated cells were harvested and homogenized in PBS on ice, the detected result was read by a microplate reader, and the glutathione in the cell lysate was calculated using the formula provided by the GSH assay kit’s product protocol.

Hepatic levels of glutathione (GSH) were measured in whole-liver homogenates (50-100 mg each of frozen liver tissue) using a glutathione assay kit (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's protocols.

4 × 10⁵ cells expressing shMLX per well were seeded into 6-well dishes and cultured for 24 hours. Then, cells were collected by trypsin digestion and prepared for measurement of glutathione using the Glutathione Assay Kit Instructions (A006-2-1, Nanjing Jiancheng) according to the user guide.