Bms223 4

The levels of IFN‐γ and TNF‐α in the cell culture medium were quantified utilizing ELISA kits (BMS228 and BMS223‐4, Invitrogen). In summary, the culture medium was collected and centrifuged at 1400 rpm for 1 min. The ELISA assay was conducted according to the provided instructions, and the absorbance at 450 nm (A450) was measured using a microplate reader (Bio‐Rad).

A transwell system with 12 inserts was used for the experiments, which contained polycarbonate filters with 0.4 μM pore sizes. Caco-2 cells were seeded in the apical (upper) chamber at a density of 2 × 10⁵ cells/chamber. The basal (lower) chamber contained 1 mL of medium. When the cells fully grow the membrane, the culture medium is removed, and the cells are incubated for 24 h with the following: 100 μM anthocyanin (AC), 1 mg/mL PT-gliadin (G), and combined treatment with AC and PT-gliadin (AC + G). Untreated culture medium served as a control. Then, supernatants from the apical chamber were collected, and human ELISA kits were used to measure the cytokine production. TNF-α (Cat.No. BMS223-4, Invitrogen), IFN-γ (Cat.No. KHC4021, Invitrogen), and IL-8 (Cat.No. BMS204-3, Invitrogen) were investigated by the supernatant according to the manufacturer’s instructions.

The levels of IFN‐γ and TNF‐α in the cell culture medium were quantified using ELISA kits (BMS228 and BMS223‐4, Invitrogen). Briefly, the culture medium was collected and centrifuged at 1400 rpm for 1 min. The ELISA assay was performed according to the kit instructions, and A450 was determined using a microplate reader (Bio‐Rad).

HCT116 and LoVo cells were seeded in 6-well plates (3 × 10⁵ cells/well) and treated as described above. At the end of treatments, media were collected and IL-8 or TNF-α protein levels measured by using specific ELISA kits (KHC0081 and BMS223-4, respectively, Invitrogen); some samples were diluted for the assay and the absorbance value was multiplied by the corresponding dilution factor, following the manufacturer’s instructions.

ELISA assays were performed for human albumin (ab108787, Abcam, Cambridge, MA USA), IL-6 (BMS213-2, Invitrogen, Seoul, South Korea), and human TNF-α (BMS223-4, Invitrogen, Seoul, South Korea) in accordance with the manufacturer’s protocol. Cell culture media were collected from the well at the given specific intervals. Culture media samples were centrifuged at 3000× g for 10 min and stored at −80 °C. The absorbance measurements were taken using SpectraMax i3 Multimode Microplate Reader (Molecular Devices, San Jose, CA, USA).

The supernatant of tumor tissues or cells was collected, and TNF-α levels in the supernatant were measured using enzyme-linked immunosorbent assay (ELISA) kits (Invitrogen, Cat#BMS223-4, Cat#88–7324-77) according to the manufactures’ instructions. To analyze the levels of TNF-α, 96-well Coat corning™ Costar™ 9018 ELISA plates were coated with diluted capture antibody overnight at 4°C. After washing in washing buffer, the wells were blocked with ELISA/ELISPOT diluent at room temperature for 1 h. Next, standards and diluted samples were added to appropriate wells, and incubated at room temperature for 2 h. After three times of washing, the wells were incubated with the detection antibody for 1 h. Diluted Streptavidin-HRP and TMB solutions were added and incubated accordingly following the instruction. After adding the stop solution, the signals were read at 450 nm.