Journal Of Biotechnology, vol.231, no.1, pp.524, 2016 (Journal Indexed in SCI)
Hydrogen peroxide (H2O2) and sulforaphane (SFN) are classical reagents to study oxidative stress and antioxidant activity in vitro, respectively. Micromole of H2O2 is sufficient to induce oxidative stress. Similarly, micromole of SFN is able to induce protective activity of cells against oxidative stress. However, higher or lower concentrations of both H2O2 and SFN may lead to aberrant results. Therefore, current study aimed to find out the appropriate concentrations of H2O2 and SFN for granulosa cells in vitro. For this, granulosa cells were collected and cultured with DMEM-F12 media at 37◦ C in a humidified environment until 60–70% confluence was achieved. After that different concentrations of H2O2 (0–400 M) and SFN (0–80 M) were added to the culture media and continue culture for 24 h. The results showed that cell viability decreased in a concentration dependent manner for both H2O2 and SFN treated cells. 50–75 MH2o2 are able to induce morphologically visible stress on cells, whereas 10–15 M SFN showed no effects on cellular morphology and viability. Analysis of candidate transcripts showed that, in H2O2 groups, 50–75 M create significantly higher expression of NRF2 & CASP3 and lower expression of KEAP1. On the other hand, 10–15 M SFN exhibited significant higher expression of NRF2 and lower expression of KEAP1, while expression of CASP3 remains unchanged. It is clear from the morphologic and genetic observations that 50–75 MH2o2 and 10–15 M SFN showed excellent response and are recommended concentrations for granulosa cells to study oxidative stress in vitro.