CellROX™ Deep Red Reagent, for oxidative stress detection

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- 1-1.5×10^5 cells were platted. - Cells were pre-treated with increasing concentrations of NS1 for 30 min at 37°C	- CellROX Reagent at a final concentration of 2 μM and incubated for 30 min.

Upstream tips
- 1-1.5×10^5 cells were platted. - Cells were pre-treated with increasing concentrations of NS1 for 30 min at 37°C
Protocol tips
- CellROX Reagent at a final concentration of 2 μM and incubated for 30 min.

Publication protocol

CellROX® Deep Red Reagent (Invitrogen) was used for oxidative stress detection (absorption and emission maxima at ~644/665 nm). HUVECs (1-1.5×105 cells) plated in 12-well plates at ~85% confluence were pre-incubated with increasing concentrations of NS1 for 30 min at 37°C, and then further incubated with 2 μM CellROX® Deep Red Reagent for 30 min. Tert-butyl hydroperoxide (TBHP) (Sigma), an oxidative stress inducer, was used as a positive control. After centrifugation at 1,000 rpm for 5 min, the cell pellets were resuspended in PBS containing 30% of enzyme-free Cell Dissociation Buffer (Gibco®) and analysed by FACS Calibur flow cytometer (Becton-Dickinson). To address the modulation effect of NS1 on ROS formation, the MFI (Mean Fluorescence Intensity) related to fluorescence emission of the CellROX® Deep Red Reagent, detected in FL-4 channel and measured in the presence of a given concentration of NS1, was normalized by the MFI obtained in the absence of NS1 (i.e. CellROX® Deep Red Reagent alone; also measured in FL-4 channel), giving the fluorescence enhancement factor. Similar measurements were performed in A375 melanoma cells.

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for CellROX™ Deep Red Reagent, for oxidative stress detection below.

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