I have used difference concentration of hydrogen peroxide to develop an oxidative stress model in Pc12 cells. The cell viability was gradually decreased as concentration of H2O2 increased ( result attached). The level of reactive oxygen species was also increased significantly in H2O2 model group (0.4mM) as compared to control group and then decrease after treating with drug/ hydrolysates (result attached).
But in case of MDA, SOD and GSH enzyme assay, there showed no difference between control and model (H2O2 induced) group. The results are totally same in control, model and drug groups.
Method used:
After complete treatment of cell culture, incubate with my samples (drug/ hydrolysates) and then incubated with H2O2. Cells were washed with medium and PBS, added RIPA buffer and placed on ice for 40 minutes, then centrifuge at 13000g* 4 degree for 10min and used supernatent for content MDA, SOD and GSH determination.
I need guidance, why the results are like this? How can i improve this? is there problem during cell lysis or something else?
Thanks in advance
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