The apoptotic rate of cells in testicular tissues collected from diabetic mice was examined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) Detection Kit (Thermo Fisher Scientific, Waltham, MA, USA). First, terminal deoxynucleotidyl transferase (TdT) reagent and dUTP reagent were mixed proportionally to prepare the working solution. Before analysis, tissue sections (4 μm) were dewaxed, permeabilized, and incubated with the working solution at 37°C for 2 hours. Sections were then washed with PBS and incubated with hydrogen peroxide solution for 15 minutes. After decolorization, sections were treated with converter-POD reagent for 30 minutes, followed by the DAB solution, and finally by hematoxylin dye for nuclear staining. Apoptosis was examined under a fluorescence microscope (Olympus).
Tunel detection kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) Detection Kit is a laboratory tool used to detect and analyze DNA fragmentation, a hallmark of apoptosis or programmed cell death. The kit provides the necessary reagents and protocols to perform this specialized assay, allowing researchers to visualize and quantify apoptotic cells within a given sample.
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3 protocols using tunel detection kit
1
Apoptosis in Diabetic Testicular Tissue
2
Quantifying Tumor Cell Apoptosis via TUNEL
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Tissue apoptosis was determined with a TUNEL Detection kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) in compliance with the manufacturer's protocol. Tumor samples from in vivo studies were rinsed in PBS and fixed in 10% paraformaldehyde/PBS for 20 min at 25°C. Samples were dehydrated in 70% ethanol, paraffin embedded, and sectioned (4-µm). The slides were rinsed twice with PBS. A total of 50 µl TUNEL reaction mixture was added on the sample and covered with parafilm during the incubation. The samples were incubated at 37°C for 1 h in a humidified chamber in the dark. Finally, the samples undergoing apoptosis were counted in three randomly chosen fields in a drop of PBS under a fluorescence microscope.
3
Cell Viability, Cycle, and Apoptosis Analysis
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Cell viability was analysed by MTS assay. 3000‐4000 cells/well were seeded into 96‐well plates. Following the indicated incubation time, media was removed and 100 μL full media with 20 μL MTS (3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3carboxymethoxyohenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium, Promega) was added. The experimental plates were incubated for another 2 hours. Then, the intensity of soluble product was measured by spectrophotometer at 490 nm. At least triplicate wells were used for each condition. Y‐axis is calculated using the following equation: day 1‐7 absorbance/day 0 absorbance and presented as folds relative to day 0. Cell cycle and apoptosis were evaluated by flow cytometry on BD FACSAria. For cell cycle analysis, the cells were fixed and stained by propidium iodide. The eBioscience™ Annexin V Apoptosis Detection Kit (Thermo Fisher Scientific) or TUNEL Detection Kit (Thermo Fisher, Scientific) were used for apoptosis assay following the standard protocol.
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