5 sulfosalicylic acid dihydrate

The treated HUVECs were harvested and lysed by sonication at 0 °C for 20 min, followed by centrifugation at 15,000 × g at 4 °C for 10 min. The cleared supernatant was prepared to detect the total protein concentration using a Protein Assay Kit (Thermo Fisher Scientific) and to measure the amount of GSH using the GSSG/GSH Quantification Kit. To remove proteins from samples, 1/6 volume of 5% 5-sulfosalicylic acid dihydrate (Wako Pure Chemical Corporation, Japan) in distilled water was used. Samples were centrifuged at 15,000 × g at 4 °C for 10 min, and the supernatant was used for the GSH assay. The protein and GSH levels in the samples were detected according to the manufacturer’s instructions by measuring the absorbance at 405 nm using a plate reader. Values for total GSH levels were calculated, corrected for protein concentration in the same sample, and normalized to the control.

Concentrations of the reduced (GSH) and oxidized (GSSG) forms of glutathione were determined with a GSSG/GSH quantification kit (Dojindo Laboratories, Kumamoto, Japan). The harvested cells were lysed in 10 mM HCl and 1% 5-sulfosalicylic acid dihydrate (Wako Pure Chemical Industries). The lysate was centrifuged (8,000×g) and the supernatant was collected. An equal volume of H₂O was added to the supernatant and the mixture was incubated with coloring agents. The tumor tissues were lysed in 5% 5-sulfosalicylic acid dihydrate and homogenized with a bead cell disrupter MS-100R (Tomy Seiko Co., Ltd., Tokyo, Japan). After the lysate was centrifuged, the supernatant was collected and added H₂O up to a final concentration of 0.5% 5-sulfosalicylic acid. The supernatant with 0.5% 5-sulfosalicylic acid was incubated with coloring agents as described above. The absorption of DTNB (λ_max = 412 nm) was measured with a multi mode plate reader PowerScan HT (DS Pharma Biomedical Co., Ltd., Osaka, Japan), and concentrations of GSH and GSSG were estimated in accordance with the manufacturer’s protocol.