2.10. Effect of C3Ghv on ROS generation in STZ treated cells

For measuring STZ-induced ROS levels, the fluorescent dye 2, 7-dichloro fluorescein diacetate (DCFH-DA) was used (Aranda et al., 2013). Briefly, RINm5F cells (2 × 10⁴ per well) suspended in RPMI-1640 medium were pre-treated for 24 h with C3Ghv (0–50 µg/mL) and further subjected to treatment with 10 mM STZ for 1 h at 37 °C. After that 20 μM DCFH-DA was added and further subjected to 30 min incubation at 37 °C. The DCFH-DA gets deacetylated by the cells and results in non-fluorescent DCFH (2′, 7′-dichlorofluorescin) production, which gets converted into fluorescent DCF (2′, 7′-dichlorofluorescein) on oxidation or reaction with ROS. The cells were then washed with phosphate-buffered saline (PBS) and homogenized in 300 μL of 0.1% Triton X-100 (PBS, pH 7.4) by sonication. The homogenates were then incubated at 5 °C for 10 min, and the supernatant was collected by centrifugation, which was used for the assay where the excitation/emission wavelength was 488/510 nm.