I have done a ROS assay using DCFDA (Sigma #D688) in a lung cancer cell line by using a microplate reader at Ex/EM 480/530. I am looking to get the quantification of the data, is there any formula to find out how much ROS has generated in treated one cell lines compared to non treated. As a positive control, I have used hydrogen peroxide.
Kindly let me know if anybody has done it.
Thank you!
If you have used complete kit from sigma, then in the data sheet there is a conversion table. You have to make a concentration curve of positive control and then you can quantify the fluorescence.
A second method could be to both use a negative control (no treatment) and just compare the values of treated cell lines to negative and positive control. You can either use absolute values or convert them into percentage.
Thank you, Sir! I haven't used the complete kit of DCFDA, just brought DCFDA catalog no D6883 (Sigma), sorry above I had missed one digit of the catalog no.
I did it by the second method, which you have mentioned here, I am going to plot a graph by using absolute value, either I could convert it into a percentage by dividing the treated sample with untreated one. I kept negative control as an unstained. For positive control, I have used 0.03% H2o2. There is some literature that suggested 0.03% of h2o2 is fine.
Please look at the following below links which may help you in your analysis:
https://www.abcam.com/ps/products/113/ab113851/documents/DCFDA-H2DCFDA-Cellular%20ROS-Assay-Kit-protocol-book-v12-ab113851%20(website).pdf
https://www.caymanchem.com/pdfs/601520.pdf
Thanks
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