Hi,

I have not used the abcam kit but have used DCFDA as such to measure ROS levels in SHSY5Y cell line and primary mouse neuronal culture too. To my experience, the analysis is straightforward. The more the DCF fluorescence, more is the ROS levels.

Hope this will help your analysis.

Alpana

Thanks, Alpana! May I ask your protocol to stain and how long do you incubate your cells in DCFDA before you measure ROS? May I also ask the conc. of DCFDA you use for SHSY5Y cells? If you could share your protocol. Highly appreciated!

Thanks again!

I have used 10 uM DCFDA and stained for 30 minutes at 37 C. Then measure ROS fluorescence using spectrofluorimeter.

Hope this helps.

Great. Thanks for the reply. It is helpful.

Hello Parveen

If this is your kit (DCFDA / H2DCFDA - Cellular ROS Assay Kit (ab113851)), then you could set the initial fluorescence at the beginning of your experiment as 1 or 100 % and all your treatments relative to this initial fluorescence signal.

If you know how many cells you have plated down and are growing per well of your microplate, then you could also calculate the fluorescence/cell.

Hope this helps

Gisela

Hi Gisela,

I plated ~30000 cells per well. Thanks for the reply.