Dcfh da kit

Predominant ROS in mitochondria was determined using MitoSOX Red mitochondrial superoxide indicator (Yeasen, Shanghai, China). Briefly, HK-2 cells were treated with strategies as previously described; cells were incubated with 5 μM MitoSOX reagent working solution at 37°C for 15 min. Cells were then treated with DAPI solution for 15 min and imaged randomly at ×200 magnification using a fluorescence microscope. Mitochondrial ROS generation was shown as the fluorescence intensity of the red color. MitoSOX-positive cell ratio was calculated in 6 randomly selected fields per sample, and each experiment was repeated in triplicate.
Intracellular accumulation of ROS was evaluated using an oxidation-sensitive DCFH-DA kit (Sigma, the United States). Briefly, HK-2 cells were treated with strategies as previously described and then incubated with DCFH-DA (10 μmol/L) in dark for 1 h. The cells were washed with DMEM/F12 culture medium and collected in PBS. The fluorescence of cells was measured by flow cytometer (Beckman, the United States), using 488 nm excitation and 525 nm emission wavelengths. Each experiment was repeated in triplicate.

A 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) kit (Sigma, Georgetown, SC, USA) was used to detect ROS levels. After exposure to OGD/R, the cells were washed with PBS and treated with DCFH-DA (20 μM) for 30 min at 37°C in the darkness. Images were collected using a confocal microscope (Leica Microsystems, Wetzlar, Germany).

We performed the ROS analysis using a DCFH-DA kit (Sigma, St. Louis, MO, USA), following standard procedure. DCFH-DA has no fluorescence and can pass freely through the cell membrane. After it enters the cells, it becomes metabolized to DCFH which cannot pass the cell membrane. The ROS in the cells can oxidize DCFH. DCF has fluorescence that is detectable and is an indicator of ROS levels. The pancreatic single cells were first suspended in the DCFH-DA working solution at about 10⁶ cells/mL and incubated in a 37 °C water bath for 20 min. During the incubation, the solution was mixed every 3–5 min. Subsequently, the cells were washed three times in the working solution, and then serum-free medium was added to stop the reaction. The fluorescence of the cells was detected by Accuri C6 (BD). The fluorescence intensity represents the relative level of ROS.