Cell based ros assay kit

Bone marrow-derived macrophages (BMDM) were isolated and cultured as described [24 ]. To stimulate BMDM, 10⁶ cells were incubated for 30 min at room temperature with 10 μg/mL F(ab′)₂ fragments of a MIP8a antibody, generated through digestion with pepsin (Sigma-Aldrich). After washing twice with RPMI 1640, goat F(ab’)₂ anti-mouse IgG (H+L) (SouthernBiotech, Birmingham, AL) was added for 2 h at 37°C to crosslink CD89. As positive controls 2 U/mL IFNγ (PeproTech, Rocky Hill, NJ) and 10 ng/mL LPS (Sigma-Aldrich, St Louis, MO) added to 10⁶ BMDM cells for 4 h at 37°C were used. The negative control lacked the cross-linking Ab. Subsequently, CD89-specific intracellular ROS production was measured by flow cytometry using a cell-based ROS assay kit (Beyotime Institute of Biotechnology, Shanghai, China) following the manufacturer’s instructions. Macrophages were trypsinized, washed with PBS, and then incubated with DCFH-DA at a final concentration of 10 μM for 30 min at 37°C. Fluorescence was analyzed on a FACSVerse (BD Pharmingen). Intracellular ROS levels were expressed as the average DCF fluorescence intensity of the cells.

Cells were seeded in triplicate wells of 6-well plates at 1 × 10⁵ cells per well and treated with cinobufagin. Cellular ROS were stained by a cell-based ROS assay kit (Beyotime, Shanghai, China) following manufacturer’s instructions. Briefly, cells were washed with PBS, and incubated with 10 μM (DCFH-DA) for 30 min at 37 °C in the dark. After washing three times in PBS, photos were taken immediately using an Olympus fluorescent microscope. The cells were then collected through trypsinization and analyzed on the BD FACS-Calibur flow cytometer (BD Biosciences, San Jose, CA, USA). Cellular ROS levels were expressed as the average 2’,7’-dichlorofluorescein fluorescence intensity. Results were mean ± SD of three independent experiments.