2.6. ROS Detection by Using DCFH-DA

2′7′-dichlorofluorescein diacetate (DCFH-DA) is lipophilic and can diffuse freely into the cell, in which it was hydrolyzed to hydrophilic DCFH, and stays inside the cell; DCFH is easily oxidated by ROS and form fluorescent DFC, the intensity of which correlates with cellular ROS levels.

In the present study, mouse 1-cell embryos were collected and separated randomly into 3 groups: (1) KSOM medium cultivation for 30 min and recultured in KSOM (for control); (2) cultured in 60 μM H₂O₂ for 30 min and recultured in KSOM (H₂O₂ group); (3) 60 μM H₂O₂ cultivation for 30 min, washed for 3 times in KSOM medium supplemented with 40 μM ICA (according to the results of the last experiment), and cultured in 40 μM ICA until detection (H₂O₂ + ICA group).

After treatment, mouse 1-cell embryos of each group were recovered at 30 h p-hCG and moved into 50 μL KSOM microdrops (15 embryos each drop) containing 20 μM DCFH-DA at 37°C for 20 min. After washing with PVP-PBS, the samples were observed under a fluorescence microscope (Nikon, Japan) at 488 nm to excite the DFC. The images of each 1-cell embryos were analyzed to obtain the gray values.

Notably, DCFH-DA stock solution (100 mM) was made by dissolving 0.02435 g DCFH-DA powder with 0.5 mL DMSO; the working solution was diluted with KSOM medium at a dilution of 1 : 5000 to 20 μM.