We lyse the cells to get the maximum possible fluorescence extracted. At this stage, you do not worry about cellular stress as the cells are dead.

You are incubating the cells with the cell-permanent fluorescent probe for a brief period so there is no need to worry about the lack of rich media content like serum, etc. You avoid any component that interfere with the fluorescence of your probe so avoid phenol red altogether.

Before you do ROS generation/level measurement, you have to make sure that the concentration you use is not toxic in cells. You need therefore at least have parallel experiment to show this. If nontoxic concentration is proven, there is no need to normalise via protein level assay. If you are dealing with a toxic concentration, you do ROS generation at early phase; before toxicity is seen – certainly not 24 h.

ROS is short-lived. 24h is definitely not appropriate.

Suggest you to go for flow cytometric detection with time course 5' through 60'.

I dilute DCFDA in DMEM w/o phenol red and add it directly into wells. Fluorescence is read after 20-30min. For normalization, I remove all medium from wells and then add lysis buffer, e.g., RIPA buffer to lyse cells for protein measurement. Good luck!

Hello Ying Wang , I am too going to perform ROS analysis on cultured cell lines using DCFDA, followed by their estimation on multi mode reader. It would be helpful to me if you could please share your protocol for the same.

Thank you in advance.

Hello Solomon Habtemariam ,

Can you please elaborate on the reasoning behind this statement? 'If you are dealing with a toxic concentration, you do ROS generation at early phase; before toxicity is seen – certainly not 24 h' . It would be really helpful!