2 7 dichlorofluorescein diacetate dcfh da solution

After PQ and ML385 treatment, cells cultured in black 96-well plates (with a transparent bottom) were aspirated and then incubated in the dark with 10 μmol/L 2′,7′-dichlorofluorescein diacetate (DCFH-DA) solution (Beyotime, China) at 37°C for 30 mins. After washing for three times by the serum-free medium, the cells were treated with 100 μL of 600 mM 2,2′-Azobis (2-methylpropionamidine) dihydrochloride (Sigma, St. Louis, MO) solution. Dynamic DCF fluorescence was measured using a microplate reader (TECAN, Männedorf, Switzerland) with excitation at 488 nm and emission at 525 nm. After subtraction of the blank, the area under the curve (AUC) for fluorescence versus time was integrated to calculate the CAA value of each sample as follows:
$\begin{array}{c}\text{CAA}\left(\text{unit}\right)=\left\{1-\frac{\int \text{SA}}{\int \text{CA}}\right\}\times 100,\end{array}$ where SA is the area of the sample and CA is the integrated area in the control curve (that generated from control cells treated with DCFH-DA and oxidant) [18 (link)].

The level of intracellular ROS was measured using a ROS detection kit (Beyotime, China). Briefly, cells from pooled samples were incubated with 2′-7′-dichlorofluorescein-diacetate (DCFH-DA) solution (Beyotime, China) in the dark at 37°C for 30 min and then were washed with PBS and analyzed using a flow cytometer (Accuri C6, USA) within 30 min. The level of ROS was expressed as the percentage of fluorescent cells (fluorescent cells/total cells × 100%) and the experiment was repeated three times.