TUNEL Apoptosis Detection Kit (Promega, Madison, WI, USA) was performed to detect apoptotic neurons. The number of TUNEL-positive cells was counted in five randomly chosen fields within each slide at 400× with an optic microscope (Olympus, Tokyo, Japan). The index of apoptosis was calculated according to the ratio of overall apoptotic cells and the total cells.
Tunel apoptosis detection kit
Manufactured by Promega
Sourced in United States
The TUNEL apoptosis detection kit is a laboratory tool designed to detect and quantify apoptosis, a form of programmed cell death. It utilizes the TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) assay, which labels the fragmented DNA that is characteristic of apoptotic cells. The kit provides the necessary reagents and protocols to perform this analysis.
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Lab products found in correlation
Laser confocal microscopy, by Olympus (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Optic microscope, by Olympus (1 mentions)
A1 spectral confocal microscope, by Nikon (1 mentions)
Hematoxylin and eosin, by Beyotime (1 mentions)
Tcs sp8 sted, by Leica (1 mentions)
Phosphate buffer, by Solarbio (1 mentions)
Penicillin streptomycin mix, by Thermo Fisher Scientific (1 mentions)
Cisplatin, by Meryer (1 mentions)
Rabbit anti γh2ax antibody, by Abcam (1 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Glacial acetic acid, by Meryer (1 mentions)
14 protocols using tunel apoptosis detection kit
1
TUNEL Assay for Apoptotic Neurons
2
Apoptosis Detection by TUNEL Assay
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TUNEL apoptosis detection kit was purchased from Promega (Madiison, WI, USA). The cells were seeded in a chamber slide and incubated overnight for attachment, and then treated with the indicated drugs. The slide was washed twice with cold PBS, and the cells were fixed with cold methanol for 10 min, and then added with equilibration buffer for 10 min. The supernatant was removed, and TdT (terminal deoxynucleotidyl transferase) incubation buffer (equilibration buffer, nucleotide mix and TdT enzyme) was added into the slide in the dark for 1 h, and the response was terminated by SSC solution. The detection of DNA fragments was observed by fluorescence microscopy under 200 magnifications.
3
Histopathological Analysis of Organ Tissues
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Immediately after intraperitoneal anesthesia and euthanasia, the mice were perfused with saline and 4 % PFA. The heart, lung, liver, spleen, and kidney were extracted and fixed in 4 % PFA for 24 h, embedded into paraffin, and subsequently sectioned into 5 μm. Sections were stained with hematoxylin and eosin (Beyotime, Beijing, China) and observed using a Nikon A1 spectral confocal microscope (Nikon, Tokyo, Japan). Dead cells in major organ sections were stained using a TUNEL Apoptosis Detection Kit (Promega).
4
Comprehensive Histological Analysis of Organs
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The experimental mice were anesthetized and then sacrificed after heart perfusion. All major organs, including the aorta, heart, liver, spleen, lung, and kidney, were extracted and fixed in 4% PFA for 24 h, and the surrounding adipose tissue was removed. Subsequently, the specimens were transferred to PBS containing 30% sucrose overnight to remove excess water. The organ samples were then embedded in OCT and sliced into 5 µm sections. ORO staining and H&E staining were carried out according to the established protocols. The lipid core area and the size of lesions were computed using ImageJ software. The histological changes in the major organs were assessed by two independent experts based on the H&E-stained specimens.
For TUNEL staining, three representative slides were selected for each mouse, and the presence of apoptotic cells in the liver tissue was evaluated using a TUNEL apoptosis detection kit (Promega,USA). The percentage of apoptotic cells was calculated based on the ratio of green fluorescence-positive cells. The cell numbers were quantified using ImageJ software.
For TUNEL staining, three representative slides were selected for each mouse, and the presence of apoptotic cells in the liver tissue was evaluated using a TUNEL apoptosis detection kit (Promega,USA). The percentage of apoptotic cells was calculated based on the ratio of green fluorescence-positive cells. The cell numbers were quantified using ImageJ software.
5
TUNEL Assay for Apoptosis Detection
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We labeled fragmented DNA with a TUNEL apoptosis detection kit (Promega). Drug-treated cells were fixed with 4% paraformaldehyde and subsequently rinsed with phosphate-buffered saline (PBS). After the fragmentation of genomic DNA, terminal deoxynucleotidyl transferase (TdT) catalyzes the addition of a green fluorescent probe (fluorescein (FITC)-labeled dUTP) to exposed 3′-OH, and then the nuclei are with DAPI prior to detection by confocal laser scanning microscopy (CLSM; Leica TCS SP8 STED). The percentage of cells undergoing apoptosis, as indicated by TUNEL-positive nuclei, was determined with ImageJ software.
6
TUNEL Assay for Apoptosis Detection
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The apoptosis was detected using TUNEL apoptosis detection kit (Promega) for T24 and 5637 cells. The cells were cultured with 20 μg/mL protease K at room temperature for 20 min and incubated with fluorescein isothiocyanate-TUNEL buffer in the dark for 1 h. Phosphate buffered saline without TdT enzymes was set as a negative control. The nuclei were labeled with Hoechst 33342, and the images were obtained under a microscope. The percentage of TUNEL positive cells was counted from at least 500 cells.
7
Apoptosis Detection in Cortical Neurons
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The sections were dewaxed and detected using a TUNEL apoptosis detection kit (Promega, Madison, USA). Briefly, the procedure involved digestion with proteinase K (without DNase), 3% H
2O
2 treatment, DNase I solution treatment, TdT enzyme reaction solution treatment, streptavidin-FITC labelled working solution treatment, POD-conjugated anti-FITC labelled working solution treatment, DAB treatment, hematoxylin counterstaining, differentiation with 1% alcohol hydrochloride, dehydration with ethanol, making transparent with xylene, and sealing with neutral resin. Finally, the number of TUNEL-positive neurons in the ipsilateral cortex was manually counted under an optical microscope.
2O
2 treatment, DNase I solution treatment, TdT enzyme reaction solution treatment, streptavidin-FITC labelled working solution treatment, POD-conjugated anti-FITC labelled working solution treatment, DAB treatment, hematoxylin counterstaining, differentiation with 1% alcohol hydrochloride, dehydration with ethanol, making transparent with xylene, and sealing with neutral resin. Finally, the number of TUNEL-positive neurons in the ipsilateral cortex was manually counted under an optical microscope.
8
Cisplatin-Loaded Alginate-Gelatin Hydrogel
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Cisplatin was purchased from Meryer (Shanghai) Chemical Technology Co., Ltd. CS and sodium alginate were purchased from Shanghai Macklin Inc. CaCl2 was purchased from Sun Chemical Technology (Shanghai) Co., Ltd. Gelatin was purchased from Shanghai Haohong Scientific Co., Ltd. Methacrylic anhydride, glacial acetic acid, and tetramethylenediamine were purchased from Meryer (Shanghai) Chemical Technology Co., Ltd. APS was purchased from Guangzhou Chemical Reagent Factory. Phosphate buffer and Duchenne Phosphate buffer were purchased from Beijing Solarbio Science & Technology Co., Ltd. PEGDA was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Rabbit anti-γH2AX antibody was purchased from Abcam Plc. 4′,6-Diamidino-2-phenylindole was bought from Sigma. Horseradish peroxidase-labeled goat anti-rabbit IgG antibody and Live & Dead Viability/Cytotoxicity Assay Kit was bought from Invitrogen Trading (Shanghai) Co., Ltd. Cell-CountingCCK-8 was bought from Dojindo Molecular Technologies (Japan). TUNEL apoptosis detection kit was bought from Promega (Beijing) Biotech Co., Ltd. Dulbecco's modified Eagle medium (DMEM), fetal bovine serum, and penicillin–streptomycin mix were purchased from Thermo Fisher Scientific Inc. All the other reagents and materials were used as received.
9
Quantifying Apoptosis in Tumor Cells
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The DNA fragmentation in apoptotic cells of tumor were determined by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP Nick-End Labeling (TUNEL) apoptosis detection kit (Promega, USA). All procedures were conducted according to the manufacture description. The stained sections were observed under fluorescence microscopy and evaluated for tumor apoptosis. Five random fields at 400× magnification were selected for quantitative analysis. We calculated the TUNEL staining positive percentage by dividing the TUNEL staining positive cell number with the whole cell number.
10
Quantification of Cellular Apoptosis
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Cellular apoptosis was detected using a TUNEL apoptosis detection kit (Promega corp., Madison, WI, USA) in accordance with the manufacturer’s instructions. The cells were stained with PI for nuclear counterstaining. Fluorescence images were obtained using a fluorescent microscope (Olympus BX63). For quantification of the positive TUNEL signal, a minimum of 500 HK-2 cells were counted in each well (n = 4), and the percentage of positively labeled cells was calculated. At least 10 regions at the corticomedullary junction in the sections from different mice of each group were determined and averaged.
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