Antioxidant peptide A

Kinase experiment:

Y79 cells are seeded at 2×105 cells/well in 12-well plates with 1000 μL of culture media and incubated at 37°C overnight. The cells are then exposed to varying dosages of Antioxidant peptide A (Pep-A) and Pep-B (10, 50, and 100 μM) in fresh medium and then incubated for 6 h. At the end of the incubation, the cells are collected and washed with ice-cold PBS twice and lysed with cell lysis buffer [0.1 M Tris/HCl, pH 7.4 containing 0.5 % Triton X-100, 5 mM β-Mercaptoethanol, 0.1 mg/mL PMSF]. Cell lysate is centrifuged at 14000×g for 5 min at 4°C. The supernatant contains a combined SOD activity from cytosol and mitochondria. The SOD activity is measured using the superoxide dismutase (SOD) activity assay kit. In brief, the cell lysate, buffer, enzyme, and the WST reagent are diluted, and the solution is added as per the protocol and incubated at 37°C for 20 min and read at 450 nm on a microplate reader. SOD activity is then calculated as a percentage of the inhibition activity of xanthine oxidase[1].

Cell experiment:

Y79 retinoblastoma and MIOM1 cells are seeded at 5×103 cells/well in 96-well plates and incubated at 37°C overnight. The cells are then treated with varying concentrations (10, 30, 60, and 100 µM) of Antioxidant peptide A (Pep-A) and Pep-B in a fresh medium and incubated for specific time periods (24 and 48 h). At the end of the incubation, 10 µL of MTT (5 mg/mL) is added to the cells with fresh medium (100 µL) and incubated at 37°C until formazan crystals are formed. The formazan crystals are dissolved in 100 µL of DMSO, and the reading is taken at 570 nm. Cell viability is calculated[1].

References:

[1]. Kalmodia S, et al. Bio-conjugation of antioxidant peptide on surface-modified gold nanoparticles: a novel approach to enhance the radical scavenging property in cancer cell. Cancer Nanotechnol. 2016;7:1.