MTT Assay for Barley Bran Immunomodulation

Based on the manufacturer's instructions, this assay was conducted using the MTT [3-(4, 5-Dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide] assay kit (Bioworld, UK). Briefly, splenocytes suspension was prepared in RPMI-1640 (supplemented with 50 U/ml streptomycin, 50-U/ml penicillin, and 10% FBS) and seeded into a 96-well culture plate at a specific concentration of a 2-x-106 cell/ml in the presence of 5-μg/ml Con A or 4-μg/ml LPS. Then, 100 μL of various concentrations (0.625–5 mg/ml) of barley bran extracts were added in an in-triplicate manner. The plate was incubated for 48 h under 5% CO₂ and humidified atmosphere of 95% air at 37°C temperature. After the incubation, each well was treated with 10 μL of MTT (5 mg/ml) solution and incubated again for 4 h. After that, it was treated with 100 μL of DMSO to dissolve the developed formazan particles, and the absorbance was measured using ELISA microplate absorbance reader at 550 nm. Results were expressed as an stimulation index compared with negative control (22 (link)).