CM-H2DCFDA (General Oxidative Stress Indicator)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Cells were cultured and treated with phorbol myristate acetate (PMA) and ionomycin and incubated for 30 min. - 5 × 10^5 were used for as ROS assay.	- 10 µM final concentration of Carboxy-H2DCFDA was used incubated for 30 min.

Upstream tips
- Cells were cultured and treated with phorbol myristate acetate (PMA) and ionomycin and incubated for 30 min. - 5 × 10^5 were used for as ROS assay.
Protocol tips
- 10 µM final concentration of Carboxy-H2DCFDA was used incubated for 30 min.

Publication protocol

After confirming that B. trimera hydroethanolic extract, quercetin and rutin at the evaluated concentrations were not cytotoxic to SK Hep-1 cells using the MTT assay, the treatments for the subsequent analyses were performed.

SK Hep-1 cells were pre-incubated for two different times (30 min or 6 h) using three different concentrations of B. trimera (10, 25 and 50 µg ml−1), rutin or quercetin (10, 25, and 50 μM). After pre-incubation, the unstimulated cells were washed and used for further assays.

In order to verify that B. trimera and positive controls were capable of inhibiting ROS in a PKC-dependent manner, we used phorbol myristate acetate (PMA) and ionomycin as pathway activators. Thus, after washing, the cells were treated with PMA (50 nM), a DAG analog and potent activator of PKC, and ionomycin (100 nM), which increases the release of calcium, and incubated for an additional 30 min. Subsequently, the cells were washed 2× in Hanks solution and used in subsequent assays. The cells (5 × 105) were resuspended in DMEM and treated as previously described. Diphenyleneiodonium chloride (DPI) (20 µM), a NADPH oxidase inhibitor, was used as a negative control during the first incubation (30 min or 6 h). Carboxy-H2DCFDA (10 µM) was added to assess ROS production in all samples and incubated for 30 min during the last incubation (in combination with PMA and Ionomycin or alone). In the final washing, the supernatant was discarded, and the cells were resuspended in 200 µL of fixing solution. The data acquisition was performed using a BD FACSCalibur™ flow cytometer and CellQuest™ software. Fluorescence images were obtained to illustrate ROS production in SK Hep-1 cells. The cells were treated as described above, fixed with paraformaldehyde and analyzed using fluorescence microscopy.

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