CM-H2DCFDA (General Oxidative Stress Indicator)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- 2.5 × 104 cells were plated in 24-well plate. - After 24h incubation, cells were pre-treated with SPIONs for 48 h at a final concentration of 200 µg Fe/mL	- After washing, cells were ytreated with DCFDA in 1xPBS at a concentration of 7 µM at at 37 °C for 45 min.

Upstream tips
- 2.5 × 104 cells were plated in 24-well plate. - After 24h incubation, cells were pre-treated with SPIONs for 48 h at a final concentration of 200 µg Fe/mL
Protocol tips
- After washing, cells were ytreated with DCFDA in 1xPBS at a concentration of 7 µM at at 37 °C for 45 min.

Publication protocol

The CM-H2DCFDA kit purchased from Invitrogen was used for the measurement of overall ROS levels. Briefly, 2.5 × 104 cells were plated in 24-well plates in duplicate. After overnight equilibration, cells were treated with tested SPIONs for 48 h at a final concentration of 200 µg Fe/mL. Prior to the end of treatment, culture media was remove and DCFDA in PBS 1× was added to each well at a final concentration of 7 µM and incubated in the dark at 37 °C for 45 min. After treatment, the fluorescence intensity of CM-H2DCFDA in cells was evaluated using a fluorescence cell imaging system (Evos®, ThermoFisher Scientific, Waltham, MA, USA). Data shown in Figure 5.

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