ROS-ID® Total ROS/Superoxide detection kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- To reconstitute Oxidative stress detection reagent it was added to anhydrous dimethylformamide	- Cells were incubated for for 1 h at 37 °C after adding detection reagent in complete media

Upstream tips
- To reconstitute Oxidative stress detection reagent it was added to anhydrous dimethylformamide
Protocol tips
- Cells were incubated for for 1 h at 37 °C after adding detection reagent in complete media

Publication protocol

ROS were detected with the Total ROS Detection Kit (ENZ-51011; Enzo Life Sciences). Oxidative stress detection reagent was reconstituted in anhydrous dimethylformamide to yield a 5 mM stock solution. Then 5 μM detection reagent in complete media was added to each well of a 96-well plate for 1 h at 37 °C. Cells were washed twice with HBSS (cellgro 21-023-CV; Corning), and toxin was added in HBSS. Mock- treated and H2O2-treated (1 mM final concentration) cells were included as controls. Fluorescence was detected using a Synergy 4 plate reader with a 488/528 filter set. The average value for each treatment was normalized to the mock-treated wells to establish relative induction.

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