H2DCFDA (DCFH-DA)

1. H2DCFDA is dissolved in DMSO to obtain a 10 mM stock solutions and further diluted before use.
2. Cells are incubated with 5 μM H2DCFDA solution in PBS in the dark for 30 min at 37°C, then harvested with 0.05% trypsin-EDTA solution, suspended in a fresh medium, and immediately analyzed with flow cytometer.
3. Along with the H2DCFDA probe, if indicated, use ROS-insensitive modification of the fluorescein dye DCFDA as a positive control. The staining procedure is the same as for the H2DCFDA。
Emission (Em)=517 Excitation (Ex)=492 517

• Cell lines

  hCECs

• Concentrations

  10 μM

• Incubation Time

  30 min

• Method

  Intracellular ROS levels in hCECs are measured by incubating the cells with the redox-sensitive fluorescent dye 2'7'-dichlorofluorescein diacetate (DCFH-DA) (10 μM) at 37℃ for 30 minutes in the dark. The cells are then detached from the culture wells using 0.25% trypsin-EDTA and washed twice using ice-cold PBS. Flow cytometry measurements are performed three times for each treatment. Mean fluorescence intensity is quantified using software. Cells that had been incubated without DCFH-DA are used as negative control. The distribution of fluorescent dichlorofluorescein (DCF) on the cell monolayer is visualized and photographed using a fluorescence microscope.