Tunel detection system

We observed the fluorescence of apoptotic cells by TUNEL assay using the fluorometric TUNEL detection system (Promega, Madison, WI, USA). MG-63 cells were seeded in six-well plates at a density of 2.5 × 10⁵ cells per well and incubated for 24 h. The cells were then treated with different concentrations of tomentosin (0, 10, 20, and 40 µM) for 24 and 48 h. Next, the cells were fixed with 4% formaldehyde for 20 min at 4 °C and permeabilized using 0.5% triton X-100 for 10 min at room temperature. Following that, the cells were treated with 50 µL TdT enzyme buffer and incubated for 1 h at 37 °C. The cell nucleus was stained using a Hoechst stain solution (Sigma-Aldrich). One microliter of Hoechst stain solution was dissolved in 2 mL of PBS. Labeled strand breaks were observed by fluorescence microscopy (CKX53; Olympus, Shinjuku, Tokyo, Japan).

The fluorescence of apoptotic cells was monitored by the fluorometric TUNEL detection system (Promega, Madison, WI, USA). MG-63 and U2OS cells were seeded in 6-well plates at a density of 2.0 × 10⁵ cells per well and incubated for 24 h. Then, the cells were treated with 6 μM of TA3 and/or 250 μM of Rg1 for 24 h. Then, the cells were fixed with 4% formaldehyde for 25 min at room temperature and permeabilized using 0.2% Triton X-100 for 5 min at room temperature. Following that, the cells were treated with 50 μL TdT reaction mixed solution and incubated at 37°C for 1-2 h. The cell nucleus was stained using a Hoechst stain solution (Invitrogen). One microliter of Hoechst stain solution was dissolved in 2 mL of PBS. Labeled strand breaks were observed by fluorescence microscopy (CKX53; Olympus, Shinjuku, Tokyo, Japan). CXP software (Beckman Coulter, CA, USA) was used to analyze the results. For Annexin V (apoptosis), PI (apoptosis), and DCF-DA (ROS), the absorbance at wavelength 520 nm, 620 nm, and 520 nm was assessed, respectively.

SNU308 cells were plated in 6-well plate (2 × 10⁵ cells/well) and incubated overnight. After incubation, cells were transfected with control, IL4Rα, or IL13Rα1 siRNA or treated with AZD1480 (0, 5, 10, and 20 µM) for 48 h. Then, cells were washed with PBS twice and fixed with 3.5~4% formaldehyde in PBS. After fixation, TUNEL assay was performed according to manufacturer’s protocol (TUNEL detection system, Promega, Madison, WI, USA). DNA damage of cell was observed by fluorescence microscopy (CKX53, Olympus).