Lipid Peroxidation Detection with Image-iT® Peroxidation Kit

Human osteosarcoma cells (U-2 OS) cells were plated on 35 mm glass bottom dishes (MatTek) and stained with 10 µM Lipid Peroxidation Sensor for 30 mins in complete growth medium at 37° C. For tocopherol treatment, the cells were pre-treated with 150 µM a-tocopherol for 30 mins. The cells were then treated with vehicle (DMSO) or 100 µM menadione or 200 µM tert-butyl hydroperoxide (TBHP) or 200 µM cumene hydroperoxide (CH) for 2 hrs at 37° C. Cells were stained with Hoechst 33342 during last 30 mins of compound incubation. The cells were then washed 3X with phosphate buffered saline (PBS) and then imaged on a Zeiss Axiovert inverted microscope using a 40x objective using filters for Hoechst, FITC and Texas Red channels. In control cells, most of the signal is in red channel when the cells are treated with ROS producing reagents like menadione, TBHP and CH, most of the signal is in green channel. Tocopherol pre-treatment decreased the lipid peroxidation in cells as seen from increased signal in red channel in tocopherol pre-treated samples.

Human osteosarcoma cells (U-2 OS) cells were plated on 35 mm glass bottom dishes (MatTek) and stained with 10 µM Lipid Peroxidation Sensor for 30 mins in complete growth medium at 37° C. For tocopherol treatment, the cells were pre-treated with 150 µM a-tocopherol for 30 mins. The cells were then treated with vehicle (DMSO) or 100 µM menadione or 200 µM tert-butyl hydroperoxide (TBHP) or 200 µM cumene hydroperoxide (CH) for 2 hrs at 37° C. Cells were stained with Hoechst 33342 during last 30 mins of compound incubation. The cells were then washed 3X with phosphate buffered saline (PBS) and then imaged on a Zeiss Axiovert inverted microscope using a 40x objective using filters for Hoechst, FITC and Texas Red channels. In control cells, most of the signal is in red channel when the cells are treated with ROS producing reagents like menadione, TBHP and CH, most of the signal is in green channel. Tocopherol pre-treatment decreased the lipid peroxidation in cells as seen from increased signal in red channel in tocopherol pre-treated samples.

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