Diosmetin (purity: 99.0%) was purchased from Nanjing Zelang Medical Technology Company (Nanjing, China). Adriamycin was a generous gift from Zhejiang Cancer Hospital (Hangzhou, China). A stock solution of Adriamycin (50 mM) and Diosmetin (50 mM) was prepared with dimethyl sulfoxide (DMSO) and stored at −20 °C. The stock solution was further diluted with the appropriate assay medium immediately before use. The final DMSO concentration did not exceed 0.2% throughout the study. Antibodies for procaspase-3, cleaved caspase-3, PARP, β-action, Bcl-2 and γ-H2AX were purchased from Santa Cruz Biotechnology (CA, USA). Secondary anti-mouse, anti-goat and anti-rabbit antibodies were purchased from Santa Cruz Biotechnology (CA, USA). The western blot detection reagent ECL was purchased from Pierce Biotechnology (Rockford, USA). The TUNEL cell apoptosis detection kit was purchased from Beyotime Institute of Biotechnology (Haimen, China). The Annexin V FITC-Propidium Iodide (PI) kit was purchased from Sigma Chemical Co. (St. Louis, MO).
Tunel cell apoptosis detection kit
Manufactured by Beyotime
Sourced in China
The TUNEL cell apoptosis detection kit is a laboratory tool used to identify and quantify apoptotic cells. It employs the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method to detect DNA fragmentation, a hallmark of apoptosis. The kit provides the necessary reagents and protocols to perform this analysis.
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Lab products found in correlation
Doxorubicin, by Selleck Chemicals (2 mentions)
γh2ax, by Santa Cruz Biotechnology (1 mentions)
Annexin 5 fitc propidium iodide kit, by Merck Group (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Bcl 2, by Santa Cruz Biotechnology (1 mentions)
Tunel solution, by Beyotime (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Solarbio (1 mentions)
Dmi3000, by Leica camera (1 mentions)
P nf κb p65, by Cell Signaling Technology (1 mentions)
Abin1, by Cell Signaling Technology (1 mentions)
Cleaved caspase 8, by Cell Signaling Technology (1 mentions)
Cck 8 kit, by Dojindo Laboratories (1 mentions)
Annexin 5 fitc and propidium iodide kit, by Keygen Biotech (1 mentions)
Nuclear and cytoplasmic protein extraction kit, by Keygen Biotech (1 mentions)
Jurkat t cells, by American Type Culture Collection (1 mentions)
Ab182981, by Abcam (1 mentions)
Ab9498, by Abcam (1 mentions)
Annexin 5 pe apoptosis detection kit, by Thermo Fisher Scientific (1 mentions)
22 protocols using tunel cell apoptosis detection kit
1
Diosmetin and Adriamycin Synergistic Apoptosis
2
Quantifying Apoptosis with TUNEL Assay
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Apoptotic cells were assessed based on the manuals of a TUNEL Cell Apoptosis Detection Kit (Beyotime). Briefly, 30-min fixing of the transfected GC cells (1 × 105) was carried out with 4% paraformaldehyde (Solarbio, p1110, Beijing, China) at 4ºC, followed by a 5-min incubation with 0.3% TritonX-100 (Sigma-Aldrich, 9002–93-1). Afterwards, TUNEL solution (Beyotime, c1088) was supplemented to each well for a 60-min incubation in dark at 37ºC. After that, DAPI (Sigma-Aldrich) was used for nuclei staining in dark at room temperature for 10 min, followed by observation and photographing under the inverted fluorescence microscope. Finally, the positive cell rate, defined as TUNEL positive cells/DAPI positive cells, was counted using Image-J software.
3
TUNEL Apoptosis Detection in FFPE Tissues
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The paraffin-embedded tumor tissue sections were mounted and baked, then the apoptotic cells were observed and calculated under the microscope with reference to the instruction of TUNEL cell apoptosis detection kit (Beyotime Institute of Biotechnology, Shanghai, China).
4
Apoptosis Quantification via TUNEL Assay
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TUNEL assay was performed as previously described (27 (link),29 (link)). Briefly, each group of cells treated as indicated was fixed with 4% paraformaldehyde, rinsed with PBS, then permeabilized with 0.1% Triton X-100 for fluorescein isothiocyanate (FITC)-end-labeling the fragmented DNA of apoptotic SH-SY5Y cells using a TUNEL cell apoptosis detection kit (Beyotime Institute of Biotechnology, Shanghai, China). The FITC-labeled TUNEL-positive cells were imaged under a fluorescent microscope (DMI3000; Leica, Allendale, NJ, USA) using 488 nm excitation and 530 nm emission.
5
Quantifying Neuronal Cell Apoptosis via TUNEL
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The measurement of neuronal cell death was carried out with TUNEL Cell Apoptosis Detection Kit (Beyotime C1091, China) following the manufacturer's instruction. Briefly, paraffinized brain tissue sections were deparaffinized and rehydrated. Subsequently digesting protein with 20.0 mg/ml proteinase K for 20 min at RT. Endogenous peroxidase was inactivated with 3% H2O2 in PBS for 20 min at RT. Labeling with biotinylated dUTP in TdT enzyme buffer incubated at 37°C for 1.0 h. After stopping the enzymatic reaction, sections were washed with PBS, covered with anti-digoxigenin peroxidase conjugate and incubated for 30 min at RT in a humid chamber. Then, sections were incubated in diaminobenzidine (DAB) until color development was achieved. Finally, sections were washed, counterstained with haematoxylin, dehydrated and mounted for microscopic detection (43 (link)).
6
TUNEL Staining for Apoptosis Detection
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TUNEL staining was performed using the TUNEL cell apoptosis detection kit (Beyotime, China) according to the manufacturer’s instructions. Briefly, sections (4 µm) were deparaffinized in xylene, rehydrated in decreasing concentrations of ethanol, boiled for 10 min, and digested in 0.5% pepsin for 60 min at 37 °C, before endogenous peroxidase was blocked in 3% hydrogen peroxide. Then put TUNEL for 1 h at 37 °C. The FITC-labeled TUNEL-positive cells were imaged under a fluorescent microscope by using 488 nm excitation and 530 nm emission. The cells with green fluorescence were defined as apoptotic cells.
7
Quantification of Tumor Cell Apoptosis
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Cell apoptosis was determined in situ with a terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) cell apoptosis detection kit (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. Briefly, 4 µm tumor sections were dewaxed with xylene twice for 5 min, and soaked in 100% ethanol for 5 min, 90% ethanol for 2 min and 70% ethanol for 2 min. Sections were rinsed with distilled water for 2 min and incubated with 20 µg/ml proteinase K without DNase at 37°C for 15 min. They were eventually washed three times with PBS, and exposed to 50 µl TUNEL working fluid. A fluorescence microscope was used to capture images of 400 high-power fields from the slides. The apoptosis index (%) was calculated according to the following formula: Apoptosis index=Number of apoptotic cells/Total number of nucleated cells ×100. Experiments were performed three times.
8
Mechanistic Insights into T. gondii Immunomodulation
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The T. gondii ME-49 strain was provided by the Laboratory of Parasitology, Jinan University. The Jurkat T-cells and Molt-4 T-cells were obtained from the American Type Culture Collection (ATCC). The rabbit monoclonal anti-human antibodies pNF-κBp65, A20, ABIN1, and Cleaved caspase-8 were from Cell Signalling Technology (Boston, USA), and the NF-κB p65, MALT1 antibody was from Abcam (Cambridge, UK). The mouse monoclonal anti-human GAPDH and secondary antibodies were purchased from Santa Cruz Biotechnology (Dallas, USA). shRNA for A20 and ABIN1 were designed and synthesised by Guangzhou OBIO Co, Ltd. (Guangzhou, China). CCK-8 kit was purchased from Dojindo (Kumamoto-ken, Japan). Nuclear and Cytoplasmic Protein Extraction Kit and Annexin V-FITC and propidium iodide (PI) kit were purchased from KeyGen Biotech (Nanjing, China). The TNIP1-3Flag-IRES2-EGFP recombinant plasmid was constructed by OBIO Co., Ltd. (Guangzhou, China). The MALT1 inhibitor MI-2 was purchased from MedChem Express (USA). The TUNEL Cell Apoptosis Detection Kit was purchased from Beyotime Biotechnology (Shanghai, China).
9
Immunohistochemical Analysis of Liver Xenografts
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The liver orthotopic xenografts were used to perform IHC staining as previously described with primary antibodies against Ki67 (#9027, 1:400, Cell Signaling Technology) or CD31 (ab182981, 1:1000, Abcam, Cambridge, MA, USA) [49 (link)]. The liver orthotopic xenografts were also used to undertake TUNEL assays with the TUNEL Cell Apoptosis Detection Kit (Beyotime, Shanghai, China). Human HCC tissues were used to carry out IHC staining with primary antibodies against CD31 (ab9498, 1 µg/ml, Abcam) as above described.
10
Apoptosis Detection in Lymphoma Cells
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Apoptosis assay was performed using Annexin V-PE Apoptosis Detection Kit (Invitrogen) and TUNEL Cell Apoptosis Detection Kit (Beyotime, Shanghai, China) according to the manufacturer’s protocol. In brief, either transfected Farage or EBV-infected SU-DHL-4 cells (4 × 105 per well) were treated with binding buffer with 5 μl Annexin V-PE and analyzed for apoptosis rates by flow cytometry (BD Biosciences, San Jose, CA, USA). Apoptotic cells were stained with 50 μL of TUNEL for 30 min at room temperature and captured under a fluorescence microscope (Leica, Wetzlar, Germany).
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