Terminal deoxynucleotidyl TUNEL assay, a method for detecting DNA fragmentation, was used to measure neuronal apoptosis. The one-step TUNEL apoptosis assay kit is obtained from KeyGEN BioTECH (KGA7074). In brief, after respective treatment, cells plated on slides were fixed with 4 % formaldehyde for 30 min in room temperature. After washed three times by PBS, slides were incubated with 1 % Triton X-100 for 5 min and then incubated with FITC-conjunctive fluorescein-12-dUTP for 30 min in 37 °C. The nuclei were stained with DAPI. Fluorescent images were acquired using a confocal microscope.
One step tunel apoptosis assay kit
Manufactured by Keygen Biotech
Sourced in China
The One Step TUNEL Apoptosis Assay Kit is a laboratory product designed to detect and quantify apoptosis, a form of programmed cell death. The kit provides a simple, one-step procedure for the detection of DNA fragmentation, a hallmark of apoptosis, using the Terminal deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) method.
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Lab products found in correlation
Fluorescence microscope, by Olympus (4 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Anti lyz, by Abcam (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Pd98059, by Merck Group (1 mentions)
3 4 5 dimethylthiazolyl 2 2 5 diphenyltetrazolium bromide mtt rnase, by Merck Group (1 mentions)
Celecoxib, by Pfizer (1 mentions)
Annexin 5 fitc and pi, by Merck Group (1 mentions)
Flow cytometry, by BD (1 mentions)
Hoechst 33258, by BD (1 mentions)
Hoechst 33258 solution, by Beyotime (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Bodipy, by Thermo Fisher Scientific (1 mentions)
2 4 isothiocyanatobenzyl diethylenetriaminepentaacetic acid p scn bn dtpa, by Macrocyclics (1 mentions)
Oxaliplatin, by Meilun (1 mentions)
Fluorescent microscope, by Olympus (1 mentions)
Fluorescence microscopy, by Olympus (1 mentions)
Cck 8 assay kit, by Dojindo Laboratories (1 mentions)
16 protocols using one step tunel apoptosis assay kit
1
Quantifying Neuronal Apoptosis via TUNEL
2
Apoptosis Detection in Kidney Cells
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Tissue sections (3 μm) were used to detect DNA fragmentation for apoptosis using the One Step TUNEL Apoptosis Assay Kit (KeyGEN Biotech, Jiangsu, China) according to the manufacturer’s instructions. Kidney cells with TRITC nuclear markers were considered TUNEL-positive. The cells were counterstained with DAPI, and fluorescent images were acquired using a confocal microscope.
3
Apoptosis Detection via TUNEL Assay
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The TUNEL staining was carried out using a One-Step TUNEL Apoptosis Assay Kit (KeyGen, China), according to the manufacturer’s instructions.
4
Apoptosis Detection in Intestinal Epithelial Cells
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Detection of apoptosis in intestinal epithelial cells was performed using the one-step TUNEL Apoptosis Assay Kit (Keygen biotech, Jiangsu, China). After baking, dewaxing, and rehydrating, paraffin sections were dipped in PBS buffer. Then, the sections were incubated with proteinase K for 30 min and TdT enzyme reaction solution for 1 h at 37 °C. The sections were labelled with streptavidin-fluorescein for 30 min at 37 ℃. Rabbit monoclonal anti-Lyz (1:250; Abcam, Cambridge, UK) was used as the primary antibody to label Paneth cells. Nuclei were counterstained with DAPI. The slides were observed under a fluorescence microscope (Nikon, Japan). TUNEL+ Paneth cells and TUNEL+ TA cells were counted by two experimenters blinded to the experimental design. At least 20 crypts were counted randomly for each slide and the mean value was calculated.
5
Synthesis and Evaluation of ZD6474, Celecoxib, and PD98059
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ZD6474 was synthesized according to a patent (number: wo2,003,039,551) procedure [31 (link)-33 (link)]. Celecoxib was purchased from Pfizer Inc. (New York, USA). PD98059 was purchased from Sigma (St. Louis, USA). All cell culture materials were purchased from Gibco-Life Technologies (Gaithersburg, MD, USA). Anti-EGFR and COX-2 antibodies were obtained from Signalway Antibody Co., Ltd (Maryland, USA) and Epitomics (Burlingame, USA). Secondary antibodies for Western blot were obtained from Santa Cruz Biotechnology (San Diego, CA). All other antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), RNase, propidium iodide (PI) were purchased from Sigma (St. Louis, USA). Annexin-V/PI binding assay kit was from Invitrogen Ltd. (Paisley, UK), One Step TUNEL Apoptosis Assay Kit was from Keygen BioTECH (Nanjing, China). Other chemicals were from Beyotime Biotechnology Company (Shanghai, China).
6
Apoptosis Quantification in Bladder Smooth Muscle
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The apoptosis level in the bladder smooth muscle was measured by the one-step TUNEL apoptosis assay kit (KeyGen Biotech, Nanjing, China) according to the manufacturer's instructions. In brief, the sections were regularly hydrated and immersed in 1% Triton X-100 followed by being incubated with proteinase K solution for 30 minutes. And the sections were reacted with TdT solution for 1 hour and then streptavidin-TRITC solution for 30 minutes in a humidified and dark chamber, respectively. Finally, DAB detection kit was used to stain the sections to observe and analyze apoptotic cells by a pathologist in a blinded manner.
7
TUNEL Assay for Apoptosis Quantification
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TUNEL assay, a method for detecting DNA fragmentation, was used to measure apoptosis. The one-step TUNEL apoptosis assay kit was obtained from KeyGEN BioTECH (KGA7074). Briefly, brain sections were rinsed in 0.5% Triton X-100 in 0.01 M PBS for 20 min at 80 °C to increase permeability of the cells. To label the damaged nuclei, 50 μl of FITC-conjunctive fluorescein-12-dUTP reaction mixture was added to each sample in a humidified chamber, which was followed by a 60-min incubation at 37 °C. The nuclei were stained with DAPI. Fluorescent images were acquired using a confocal microscope.
8
Quantifying Apoptosis via TUNEL Assay
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TUNEL staining for apoptotic cells was performed using One-Step TUNEL Apoptosis Assay kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) according to the manufacturer's protocol. Nuclei were stained with DAPI. Fluorescence signals were detected with a fluorescence microscope (Olympus Corporation, Tokyo, Japan). Apoptosis index was quantified as the ratio of (number of TUNEL-positive cells)/(number of total cells) ×100%.
9
Quantification of Neuronal Apoptosis
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Cellular apoptosis was quantified via TUNEL staining following the protocol of the One Step TUNEL Apoptosis Assay Kit (Keygen, Nanjing, China). The treated N2a cells and MCAO brain slices were stained according to the manufacturer’s instructions [36 (link)]. The survival rates of the neurons in the mouse penumbra were evaluated via Nissl staining. Briefly, the brain slices were stained with Nissl staining solution at 37°C for 10 min. Similarly, TUNEL-positive cells and typical Nissl bodies were counted under a fluorescence microscope (Olympus, Tokyo, Japan).
10
Apoptosis Evaluation in SGC7901/DDP Cells
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SGC7901/DDP cells treated under different conditions as indicated were harvested in PBS. Then, the cells were incubated with Annexin V-FITC and PI (Sigma-Aldrich) for 15 min in a darkroom at room temperature. Then, the stained cells were analyzed through flow cytometry (BD Pharmingen).
Hoechst 33258 staining of SGC7901/DDP cells was used to determine the apoptotic status. Cells (1×106)/ml SGC7901/DDP cells were seeded in 6-well plates and then treated as indicated. The cells were collected after various treatments, washed with PBS twice, and fixed with 4% paraformaldehyde (Taize Ruida Technology Co. Ltd., Beijing, China) for 10 min and then washed with PBS three times. Next, the cells were stained with Hoechst 33258 solution (Beyotime) following the manufacturer's instructions. The representative images were immediately captured through a fluorescence microscope (Olympus Corp., Japan) to calculate the apoptotic cells.
SGC7901/DDP cells were exposed to LIQ, DDP or the two in combination for 24 h on 12-well plates. Next, the One Step TUNEL Apoptosis assay kit (KeyGen Biotech) was used to determine the apoptosis following the manufacturer's instructions. The staining intensity was assessed through a fluorescence microscopy.
Hoechst 33258 staining of SGC7901/DDP cells was used to determine the apoptotic status. Cells (1×106)/ml SGC7901/DDP cells were seeded in 6-well plates and then treated as indicated. The cells were collected after various treatments, washed with PBS twice, and fixed with 4% paraformaldehyde (Taize Ruida Technology Co. Ltd., Beijing, China) for 10 min and then washed with PBS three times. Next, the cells were stained with Hoechst 33258 solution (Beyotime) following the manufacturer's instructions. The representative images were immediately captured through a fluorescence microscope (Olympus Corp., Japan) to calculate the apoptotic cells.
SGC7901/DDP cells were exposed to LIQ, DDP or the two in combination for 24 h on 12-well plates. Next, the One Step TUNEL Apoptosis assay kit (KeyGen Biotech) was used to determine the apoptosis following the manufacturer's instructions. The staining intensity was assessed through a fluorescence microscopy.
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