I have been treating HUVEC with my compound to see if it can reduce the TNF-a induced ROS. Usually I treat the compound twice (with DCFDA and after washing with TNF-a). Since the results were not satisfactory, I treated differently
1. Treat the compound for 1hr before DCFDA
2. After DCFDA (compound + TNF-a; 1hr)
From no. 1 I got a little reduction with compound in comparison to TNF-a (+) group, but no. 2 all the groups went up above the TNF-a(+) group.
The compound I am using is a tyrosine kinase inhibitor.
From these results what would the best speculation about the compound action?
Please share your ideas and suggestion.
Thanks in advance.
I did not follow your question 100%, but I would suggest that you pre-treat your cells with your compound and stimulate TNF-a induced ROS. Thereafter you load the DCFDA (which is specific for H2O2). Remember to wash your sample prior to measuring.
From your results (No 1 experiment)it is evident that your compound ameliorated the generation of ROS and I will stick to that.
Dear Stefan,
Thank you for your insightful answer. I'll try that for sure.
