Glutathione fluorescent detection kit

The capability in modulating immune systems was evaluated based on the release of pro-inflammatory cytokines IL-8 in Caco-2 cells during hydrogen peroxide (H₂O₂)-induced oxidative stress. IL-8 release in supernatants were measured by a Human IL-8/CXCL8 ELISA Kit as per the manufacture’s instruction (Sigma-Aldrich, Cat #RAB0319-1KT). Total glutathione was measured to evaluate the intrinsic cellular antioxidant responses during the anthocyanin-rich treatments [34 (link)]. Post-treatment Caco-2 cells were washed with cold PBS three times and immersed in cold PBS before being collected by scraping method using Falcon™ Cell Scrapers. The collected samples were centrifuged at 600× g for 5 min at 4 °C and the pellets were suspended in 500 µL of ice cold 5% aqueous 5-sulfosalicylic acid dihydrate (SSA). Cells were disrupted by sonicating in ice-water bath for 5 min, followed by an incubation at 4 °C for 10 min. Subsequently, samples were centrifuged at 14,000× g for 10 min at 4 °C, and the supernatants were collected and stored at −80 °C for further analysis. The total GSH was measured by a Glutathione Fluorescent Detection Kit (Invitrogen, Cat #EIAGSHF) following the manufacturer’s instructions.

A glutathione fluorescent detection kit (Invitrogen GmbH, Karlsruhe, Germany) was performed to analyze the amount of free glutathione (GSH) in W1 and W1CR cells. For this, cell lysates were made as already explained above with different treatments. A Pierce™ BCA protein assay kit was used to quantify total protein. The assay was performed according to the manufacturer’s instructions. After incubation at room temperature, the 96-well plate was measured in a FLUOstar Omega Fluorescence (BMG Labtech) at 510 nm with an excitation of 390 nm.