Fluorescein dutp

The cells were seeded in 96-well plates at a density of 8×10³ cells/well and incubated overnight. After treatment, MTT (5 mg/ml) was added to each well and incubated for 4 h, and then supernatants were removed. The cells were solubilized in 200 µl dimethyl sulfoxide (DMSO) and the absorbance was recorded with a microplate reader at the wavelength of 490 nm. The TUNEL assay was used to detect apoptosis. A one-step TUNEL kit (Beyotime Institute of Biotechnology) was used for TUNEL staining, according to the manufacturer's protocol. The cells were incubated with fluorescein-dUTP (Invitrogen; Thermo Fisher Scientific, Inc.) to stain apoptotic cell nuclei and with DAPI (5 mg/ml) to stain all cell nuclei at room temperature for 3 min. The number of TUNEL-positive cells was calculated by counting at least 5 random separate fields as the ratio of the experimental samples to the control samples. A caspase-3 activity kit (Beyotime Institute of Biotechnology) was used to measure caspase-3 activity according to the manufacturer's instructions.

A TUNEL assay was performed using a one-step TUNEL kit (Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer's instructions. TUNEL staining was performed with fluorescein-dUTP (Invitrogen; Thermo Fisher Scientific, Inc.) to stain apoptotic cell nuclei, and DAPI (5 mg/ml) was used to stain all cell nuclei at room temperature for 3 min. Cells in which the nucleus was stained with fluorescein-dUTP were defined as TUNEL positive. The slides were then imaged under a confocal microscope (26 (link)).

Cellular viability detection and apoptosis assays were measured via MTT assays and TUNEL staining [45] (link). The MMT assay was conducted as previously described. Briefly, the cells were seeded in a 96-well plate at 37 °C with 5% CO₂. Subsequently, 20 µl 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mg/ml; pH 7.4; Sigma-Aldrich) was added to the cells for 4 h. The supernatants were then discarded, and 100 µl dimethyl sulfoxide (Sigma-Aldrich) was added to each well for 10 min. The OD of the samples was measured at an absorbance of 490 nm using a spectrophotometer (Epoch 2; BioTek Instruments, Inc., Winooski, VT, USA). The assay was repeated 3 times.
A TUNEL assay was performed using a one-step TUNEL kit (Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer's instructions. TUNEL staining was performed with fluorescein-dUTP (Invitrogen; Thermo Fisher Scientific, Inc.) to stain apoptotic cell nuclei, and DAPI (5 mg/ml) was used to stain all cell nuclei at room temperature for 3 min. The cells in which the nucleus was stained with fluorescein-dUTP were defined as TUNEL positive. The slides were then imaged under a confocal microscope.