Reactive oxygen species (ROS) production in HK-2 cells in response to HG treatment was detected as described previously.11 (link) The HK-2 cells were plated on six-well plates at 5×104 cells per well, cultured overnight, and then pretreated with or without NHE1/PKG inhibitor before being incubated with 30 mM HG. DMSO and N-(4-hydroxyphenyl) retinamide (4HPR) (Sigma-Aldrich, St. Louis, MO, USA) were used as controls. After 48 h of incubation, the cells were harvested, washed in PBS, and then loaded with 50 µM 2',7'-dichlorofluorescein-diacetate (DCFH-DA) at 37℃ for 10 min. Fluorescence was detected using a FACSCanto cytometer (BD) on the FL1 channel and analyzed with FACSDiva 6.0 software.
Protocol detail
Quantifying ROS in HK-2 cells under HG
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Corresponding Organization :
Other organizations : Union Hospital, Air Force Medical University
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Variable analysis
independent variables
- HG treatment
- NHE1/PKG inhibitor
- Reactive oxygen species (ROS) production in HK-2 cells
- Cell density (5×10^4 cells per well)
- Cell culture duration (overnight)
- Positive control: DMSO
- Positive control: N-(4-hydroxyphenyl) retinamide (4HPR)
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