Deadend fluorometric tunel system assay kit

Apoptotic DNA fragmentation was examined using an in situ DeadEnd™ Fluorometric TUNEL System assay kit (Promega, Madison, WI, USA) according to the manufacturer's protocol. Briefly, cells were plated in 24-well flat-bottom plates and treated with cisplatin (10 μM) for 36 h. Cells were fixed in 4% paraformaldehyde at 4°C for 30 min, permeabilised in 0.1% Triton X-100, and labelled with fluorescein-12-dUTP using terminal deoxynucleotidyl transferase. The localised green fluorescence of apoptotic cells from the fluorescein-12-dUTP was detected by fluorescence microscopy (Axiovert 100M, Zeiss, Oberkochen, Germany).

TUNEL assay was performed using the Dead End™ fluorometric TUNEL assay kit. The TUNEL assay for in situ detection of apoptosis was performed by using the Dead End™ Fluorometric TUNEL System assay kit (Promega, Madison, WI, USA), according to the manufacturer’s instructions. H460 and A549 cells were treated with D1399 at concentrations of 0.5 × IC₅₀, 1.0 × IC₅₀, and 1.5 × IC₅₀ for 24 h. Following, cells were fixed in 4% paraformaldehyde solution at 4 °C for 25 min. Fixed cells were washed three times with 1 × PBS for 5 min and then permeabilized in 0.1% Triton X-100 for 15 min. After rinsing the slides with 1 × PBS, cells equilibrated in an equilibration buffer for 10 min and treated terminal deoxynucleotidyl transferase (TdT) was stored away from light for 1 h at 37 °C to label with fluorescein-12-dUTP. After a wash with 2 × SSC for 15 min at room temperature, the cells were treated with 1 mg·mL⁻¹ DAPI solution for 15 min. The slides were observed by fluorescence microscopy with an inverted microscope (Zeiss Axiovert100M, Carl Zeiss, Germany). A total of ten randomly chosen microscopic fields, including green fluorescence of apoptotic cells, were captured and calculated. Experiments were performed in triplicate.

Apoptotic DNA fragmentation was examined using an in situ DeadEnd™ Fluorometric TUNEL System assay kit (Promega, Madison, WI, USA) according to the manufacturer's protocol. Briefly, cells were plated in 24-well flat-bottom plates and treated with gemcitabine (5 μM) for 36 h. Cells were fixed in 4% paraformaldehyde at 4°C for 30 min, permeabilised in 0.1% Triton X-100, and labelled with fluorescein-12-dUTP using terminal deoxynucleotidyl transferase. The localised green fluorescence of apoptotic cells from the fluorescein-12-dUTP was detected by fluorescence microscopy (Axiovert 100M, Zeiss, Oberkochen, Germany).