To perform the TUNEL assay, cells were fixed in 4% paraformaldehyde at room temperature for 30 min [29 (link)]. After that, a TUNEL kit (Roche Apoptosis Detection Kit, Roche, Mannheim, Germany) was used on the slices according to the instructions [30 (link)]. Finally, the sections were amplified to 400×; the apoptotic cells in at least 10 fields were randomly chosen. The apoptotic index was the proportion of apoptotic cells to total cells according to a previous study [31 (link)].
Apoptosis detection kit
Manufactured by Roche
Sourced in United States, Germany, Switzerland
The Apoptosis detection kit is a laboratory tool designed to detect and analyze apoptosis, a form of programmed cell death. The kit contains reagents and protocols to assess the presence and extent of apoptosis in cell samples. It provides a standardized approach for researchers to study this important cellular process.
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Lab products found in correlation
Fluorescence microscope, by Olympus (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Epoch 2, by Agilent Technologies (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
4 6 diamidine 2 phenylindole dihydrochloride dapi, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Fluorescent mounting media, by Solarbio (1 mentions)
Tunel regent, by Beyotime (1 mentions)
Proteinase k, by Beyotime (1 mentions)
Mouse anti neun antibody, by Abcam (1 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
Cell counter, by Olympus (1 mentions)
Fluorescence microscopy, by Leica Microsystems (1 mentions)
Bright field microscope, by Olympus (1 mentions)
Image pro plus v6, by Media Cybernetics (1 mentions)
Fluorescence microscope, by Leica camera (1 mentions)
Dm500, by Leica camera (1 mentions)
Alcian blue, by Merck Group (1 mentions)
Brdu cell proliferation kit, by Roche (1 mentions)
87 protocols using apoptosis detection kit
1
Quantifying Apoptotic Cells via TUNEL Assay
2
Assessing Cell Death in Liver Cancer Cells
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Cell death was measured by TUNEL staining, PI staining and MTT assays. In the TUNEL assays, cells (HepG2 cells, Huh7 cells and Hep3B cells) were fixed in paraformaldehyde at room temperature for 15 min. Then, cells were washed three times in cold PBS and treated with 0.05% Triton X-100 for 15 min on ice [47 (link)]. Subsequently, a TUNEL kit (Roche Apoptosis Detection Kit, Roche, Mannheim, Germany) was used to stain cells, as described in a previous study [48 (link)]. For the PI staining assays, HepG2 cells were washed three times with cold PBS, and PI (1 mM) was added to the medium. The cells were cultured in the dark at 37 °C, 5% CO2, for 15 min. Finally, at least 10 fields were randomly chosen under a digital microscope, and the apoptotic cells in these fields were quantified [49 (link)]. MTT assays were carried out according to a previous report [50 (link)]. Briefly, HepG2 cells were seeded into 96-well plates. Cells were treated with MTT (2 mg/mL; Sigma-Aldrich) for 4 h at 37 °C. Then, cells were washed with PBS to remove free MTT, and DMSO was added to the wells. Finally, the OD of each well was recorded, and cellular viability was calculated as described in a previous study [51 (link)].
3
Assessing Cell Viability and Apoptosis
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MTT assay was used to observe the cellular viability. Cells were seeded onto a 96-well plate, and the MTT was then added to the medium (2 mg/ml; Sigma-Aldrich). Subsequently, the cells were cultured in the dark for 4 h, and DMSO was added to the medium. The OD of each well was observed at A490 nm via a spectrophotometer (Epoch 2; BioTek Instruments, Inc., Winooski, VT, USA) [36 (link)]. TUNEL assay, cells were fixed in 4% paraformaldehyde at room temperature for 30 min. After that, a TUNEL kit (Roche Apoptosis Detection Kit, Roche, Mannheim, Germany) was used on the slices according to the instructions. Finally, the sections were amplified to 400×; the apoptotic cells in at least 10 fields were randomly chosen. The apoptotic index was the proportion of apoptotic cells to total cells according to a previous study [37 ].
4
Histological and Apoptosis Analysis of Tumor Tissue
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For histologic analysis, tumor tissue was fixed in 4% formaldehyde, dehydrated with an ethanol gradient, and embedded in paraffin. Then the paraffin tumor tissue sections (4 µm) were obtained and stained with H&E. The TUNEL assay for apoptosis analysis was performed using an apoptosis detection kit (Roche Diagnostics, Branchburg, NJ, USA) according to the manufacturer's instructions. Positive cells were identified, counted (six random fields per slides), and analyzed by light microscopy (BX51; Olympus Co.).
5
Apoptosis Quantification in Liver Tissue
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Tissues were immersed in 4% paraformaldehyde in PBS and fixed overnight at 4 °C. Fixed tissues were paraffin-embedded, cut into 3 μm thick transverse sections, and mounted on slides. An apoptosis detection kit (Roche, 11,684,817,910, Mannheim, Germany) was used according to the manufacturer’s instructions. Sections were visualized with 3, 3-diaminobenzidine tetrahydrochloride and counterstained with Gill’s hematoxylin. Cells were counted from randomly selected high-power fields (200×) from each section under light microscopy, and the rates of TUNEL-positive cells were calculated. A total of 500 hepatocytes from each mice were used to count positively stained cells.
6
TUNEL Staining for Neuronal Apoptosis
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TUNEL staining was carried out on the cerebral sections using the apoptosis detection kit (Indianapolis, Roche, IN). Staining of sections was performed according to the manufacturer’s instructions. Afterward, cerebral sections were incubated with DAB (3, 3’-diaminobenzidine) and hydrogen peroxide; the brownish color shows damage to neuronal perikarya (Hakemi, Sharififar, Haghpanah, Babaee, & Eftekhar-Vaghefi, 2019 (link); Varshosaz, Taymouri, Pardakhty, Asadi-Shekaari, & Babaee, 2014 (link)). Also, negative and positive controls were included.
7
TUNEL Assay for Apoptosis Evaluation
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Cell apoptosis rate in ARPE-19 cells and rat retinal tissues was visualized by using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay with an apoptosis detection kit (Roche, Mannheim, Germany). The TUNEL assay was performed according to the manufacturer's protocol.
8
Apoptosis Detection in Brain Tissue
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Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to determine apoptosis in the brain tissue infarct area using an apoptosis detection kit (Roche Applied Sciences, Penzberg, Germany). The assay was conducted according to the manufacturer’s instructions [77 (link)]. Briefly, all samples were cut using cryo-section and fixed with 4% PFA for 15 min at room temperature. The tissue sections were permeabilized and incubated with the TUNEL reaction mixture including TdT and fluorescence-labeled dUTP at room temperature. Nuclei were stained with DAPI. The apoptotic cells were analyzed with a confocal microscope. TUNEL-positive cells were counted using Image J software version 1.54d.
9
Detecting Apoptosis with TUNEL Assay
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The TUNEL assay for the in situ detection of apoptosis was performed using an apoptosis detection kit (Roche, California, USA) following the manufacturer's instructions.Brie y, cells were rst xed with 4 % paraformaldehyde for 30 minutes at room temperature, washed twice with PBS, and then permeabilized with 0.1 % Triton X-100. The cells were then xed in 20 μg / ml protease K for 20 minutes, washed twice with PBS, and then stained in sequence with TUNEL reaction mixture.TUNEL positive cells are brown in nucleus. In order to show other normal nuclei, we used hematoxylin to re-dye the nuclei for counting.
10
TUNEL Staining for Apoptosis Detection
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TUNEL staining was performed with an apoptosis detection kit (Roche Applied Science, Indianapolis, IN, USA). Tissue sections were dewaxed and digested using protease K. Samples in test groups were added with 50 μl of TUNEL reaction mixture and incubated at 37°C for 60 min in the dark and further dried. Then the sections were incubated with 50 μl of converter-POD for 30 min at 37°C, washed with PBS, and developed at room temperature with the DAB color solution (Wuhan Boster Biological Engineering Co., Ltd., P.R. China). Color development was terminated by rinsing with water. Harris hematoxylin restaining was performed, followed by dehydration and permeabilization, air drying, and mounting with neutral gum. Dried sections were observed, and images were acquired under a Nikon E100 biological microscope (Nikon, Tokyo, Japan). For TUNEL staining, six sections with four high-power fields each per group were observed for the assessment of cell apoptosis. Under a microscope, apoptotic cells were colored brown, and quantitative analysis of the cell apoptotic index in HCC was performed using an image analysis software, Image Pro Plus 6.0 (Media Cybernetics, Rockville, MD, USA).
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