In Vitro ROS/RNS Assay

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Cells were cultured in 96 well plate	- Cells were treated with fluorogenic probe 2′,7′-dichlorodihydrofluorescin diacetate and incubated for 1h

Upstream tips
- Cells were cultured in 96 well plate
Protocol tips
- Cells were treated with fluorogenic probe 2′,7′-dichlorodihydrofluorescin diacetate and incubated for 1h

Publication protocol

The ROS assay was performed using the OxiSelect Intracellular ROS Assay Kit (STA-342; Cell Biolabs, San Diego, CA). Briefly, Pml+/+ and Pml−/− MEFs, PML-knockdown HUVECs, or transfected HeLa cells were cultured in a 96-well plate. Cell-permeable fluorogenic probe 2′,7′-dichlorodihydrofluorescin diacetate was added to wells, followed by 1-h incubation in a cell incubator. The assay was terminated by adding cell lysis buffer and incubating for 5 min. Fluorescence intensities were quantified by a SpectraMax M2 plate reader (Molecular Devices, Sunnyvale, CA) at 480/530 nm with 530-nm cutoff. All samples were prepared in triplicate and data are presented as the mean ± SD from three experiments. Unpaired two-tail t tests were performed to determine significance.

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