Click it tunel alexa fluor 488 imaging assay kit

TUNEL assay followed by H33258 staining of nuclei was performed in HepG2 and HK-2 cells cultured in 200 µL of appropriate cell culture medium on cell culture chamber slides at density of 1.5 × 10⁵ and 2 × 10⁵ cells per well, respectively. After seeding, the culture medium was replaced by 200 µL of CisPt solutions and the cells were treated for 6, 24 and 48 h. TUNEL assay was performed using Click-iT TUNEL Alexa Fluor 488 Imaging Assay kit (ThermoFisher Scientific, USA) according to manufacturer´s instructions. The cells were fixed with 12% formaldehyde for 15 min at 37 °C. Then, the cells were permeabilized with 0.2% Triton X-100 for 15 min at 37 °C, washed with PBS 1 × and incubated with terminal deoxynucleotidyl transferase (TdT) buffer for 10 min at 37 °C. After incubation, cells were mixed with a TdT reaction mixture (TdT buffer, 5-Ethynyl-2′-deoxyuridine 5′-triphosphate, TdT) and incubated for 1 h at 37 °C. Then, the cells were washed with 3% bovine serum albumin (BSA) and Click IT reagent for fluorescent staining was added for 30 min at 37 °C. After PBS 1 × washing, H33258 at a final concentration of 2 µg/mL was used to visualize the cell nuclei. DNA strand breaks (FITC filter, 480/30 nm) and cell nuclei (DAPI filter, 375/28 nm) were visualized with an Eclipse 80i fluorescence microscope (Nikon, Japan).

DNA fragmentation was detected using the terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay (Click-iT TUNEL Alexa Fluor 488 imaging assay kit, Thermo Fisher Scientific, Cat# C10245). DLD1 cells were seeded in 8-well chamber slides (2 × 10⁴ cells/well), cultured overnight (37 °C, 5% CO₂) and incubated with 7, 21 or 35 µM BAMLET for one hour in serum-free RPMI-1640 medium at 37 °C. Cells were fixed (2% PFA, 15 min), permeabilized (0.25% Triton X-100 in PBS, 20 min) and incubated with TUNEL reaction mixture containing TdT for 60 min at 37 °C. After the TUNEL reaction, the cells were incubated with Click-iT reaction mixture for 30 min. Cells were counterstained with Hoechst 33,342 (1:1,000, 15 min, Thermo Fisher Scientific, Cat# 62,249), mounted in Fluoromount aqueous mounting medium (Sigma-Aldrich, Cat# 4680), and examined with an LSM 900 confocal microscope (Carl Zeiss). Fluorescence intensities were quantified by ImageJ.

Terminal DNA breakpoints in situ 3 - hydroxy end labeling (TUNEL) was to quantify levels of apoptosis in NHDF cells. Cells were seeded at 1 × 10⁴ cells/cm² in 6 well plates and after 10 days were treated with 5 μM of each compound for 24 h in 3 biological replicates. The TUNEL assay was performed with Click-iT® TUNEL Alexa Fluor® 488 Imaging Assay kit (Thermofisher, UK) following the manufacturer’s instructions. Negative and positive (DNase1) controls were also performed. The apoptotic index was determined by counting the percentage of positive cells from at least 400 nuclei from each biological replicate at 400× magnification.

TUNEL staining was performed on colonic sections using the Click-iT TUNEL Alexa Fluor 488 Imaging Assay kit (ThermoFischer Scientific, Waltham, USA) according to the manufacturer's instructions. Samples were evaluated using confocal microscopy (Zeiss 880, ZEN software, Zeiss, Germany). The number of TUNEL-positive cells was counted in 10 randomly chosen representative high-power fields and images were acquired using a 20X objective and 40X objective.

Cells were fixed with 4% paraformaldehyde, and immunocytochemistry was performed as described previously32 (link). For the TUNEL assay, cells were stained using the Click-iT TUNEL Alexa Fluor 488 imaging assay kit (Life Technologies) according to manufacturer instructions. Nuclei were stained with Hoechst 33258 (Sigma-Aldrich). Samples were imaged by ImageXpress (Molecular Device, Sunnyvale, CA, USA) with MetaXpress and AcuityXpress software (Molecular Device).