ROS (Reactive Oxygen Species), observing cell changes using active oxygen

ROS is known to play an important role in cells, such as cell signaling, induction, cell death, and gene expression.

There are various experimental methods using ROS, but among them, the method of measuring using a fluorescent probe is widely used.

This time, we will introduce a method of measuring and analyzing the results of ROS generation by cell growth stimulation using DCF fluorescent material with SynergyTM HT.

Among various enzymes, NADPH oxidase recognizes a portion of ROS and collects various proteins to transmit signals into the cell.

DCF reactions

To confirm the formation of ROS, fluorescence called DCF is measured. DCF penetrates the cell membrane and fluoresces strongly when it meets ROS.
If ROS production continues under a given intracellular stimulus, the fluorescence signal also continues.
On the other hand, if there is no ROS formation, fluorescence is not measured.

This is the result of measuring DCF fluorescence according to the degree of cell stimulation using SynergyHT.
PMA and Serum were used to stimulate cells, and as a positive control, an enzyme called Glutathion oxidase, which can form ROS without any special cell stimulation, was used.
Cells stimulated by these substances form ROS and then emit DCF fluorescence.
On the other hand, cells without stimulation show no change in fluorescence value.

DCF Fluorescence in Stimulated Cells Measured over Time.

Cells were treated with 1 mU/mL of Glutathione oxidase (magenta); 10 μM PMA (green); 0.25% FBS serum (red); or untreated (blue).

PMA & serum induction of H2O2 as measured by endpoint fluorescence after 30 min.

Here are the results of comparing the fluorescence measurements 30 minutes after cell stimulation. With 0% FBS, where there was no cell stimulation at all, there was almost no fluorescence measurement. However, with 0.25%, a 3-fold higher fluorescence value was measured, and with 1 μM PMA, a 5-fold higher fluorescence value was measured.