Total gsh quantification kit

Total GSH concentration was evaluated using a Total GSH Quantification Kit (Cat No. T419; Dojindo), following the manufacturer’s instructions. Harvested cells and tumor tissue were lysed in 10 mM HCl and 1% 5-sulfosalicylic acid dihydrate (Wako Pure Chemical Industries). Lysate was centrifuged (8,000×g) and the supernatant collected. An equal volume of H₂O was added to the supernatant and incubated with coloring reagents.

The Invader UGT1A1 Molecular Assay Kit (Third Wave Technologies, Madison, WT, USA) was used to detect UGT1A1*6 and UGT1A1*28 polymorphisms of genomic DNA derived from blood samples. Regarding cell line samples, the polymerase chain reaction (PCR) was carried out to amplify the characteristic regions using designed primers (Greiner Bio-One, Tokyo, Japan), and UGT1A1 polymorphisms were determined by direct sequencing, as previously described [16 (link)].
Primers: UGT1A1*28 forward: 5’-TATA GTCACGTGACACAGTC’-3 and reverse: 5’-CCACTGGGATCAACAGTATCT’-3, UGT1A1*6 forward: 5’-AAGTAGGAGAGGGCGAACC’-3 and reverse: 3’-GTGGGCAGAACAGGTACT’-3.
Total GSH concentrations in cancer cells were assayed using the total GSH Quantification Kit (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s protocol.