Gsh gssg gsh assay kit

The concentrations of total glutathione (GSH + GSSG) and GSH were assessed using a colorimetric assay (GSH + GSSG/GSH Assay Kit; Abcam, ab239709, Cambridge, MA, USA) following the manufacturer’s instructions. In this assay, cells were collected by centrifugation at 700× g for 5 min at 4 °C. Supernatant was removed and the cell pellet was resuspended in 0.5 mL ice-cold phosphate-buffered saline and centrifuged at 700× g for 5 min at 4 °C. Later, the supernatant was removed and cells were lysed in 80 μL ice-cold Glutathione Buffer and incubated on ice for 10 min. Subsequently, 20 μL of 5-sulfosalicylic acid (5%) was added and the samples were centrifuged at 8000× g for 10 min. The supernatant was transferred to a tube and used for the glutathione assay. The concentration of GSSG was determined by calculating the difference between the total glutathione and GSH values.

PGE₂ level in supernatant from expanded TILs was determined using a human PGE₂ ELISA Kit (abcam, ab287802) according to the manufacturer’s instructions. For the solid tumour cohort, PGE₂ levels were measured by ELISA during the first medium change. For the breast and melanoma cohort PGE₂ levels were measured between day 7 and 10. Total and reduced glutathione levels were determined with the GSH + GSSG/GSH Assay Kit (Colorimetric) (abcam, ab239709). Intracellular ATP levels were quantified with ATP Detection Assay Kit (ab113849). Intracellular MDA levels were measured with the TBARS fluorometric microplate assay (FR45, Oxford Biomedical Research).

The antioxidant biomarkers SOD and GSH were investigated using a colorimetric SOD activity assay kit (Sigma-Aldrich, USA) and a GSH + GSSG/GSH assay kit (Abcam, USA) according to the manufacturer's instructions, respectively.

The level of cellular glutathione including the reduced (GSH) form and total glutathione were measured by GSH+GSSG/GSH assay kit (colorimetric) from Abcam (UK). Briefly, the HT-29 and HCT-116 cells were seeded in 96-well plates and incubated at 37 ˚C in a humidified atmosphere in a 5% CO₂ incubator for 24 h. After incubation, the culture media were removed and replaced with the media containing the sample and then incubated at 37 ˚C in a humidified atmosphere in a 5% CO₂ incubator for 24 h. Next, cells were added to the reaction mix and incubated at room temperature for 10 min to generate NADPH. For detecting GSH only, glutathione reductase was omitted from the reaction mix. Afterwards, cells were added to the GSH standard or the sample solution and the plate was incubated at room temperature for 5-10 min; then the substrate solution was added to the pate and incubated at room temperature for 5-10 min. The result was determined by using an automate microplate reader (1420 Victor 2, Wallac, USA) at 405 nm.
Total glutathione = [(O.D. samples - O.D. blank) / Slope STD Curve