The TUNEL apoptosis assay kit (40307ES20, Yeasen Biotechnology Co, Ltd. Shanghai, China) was used to examine apoptosis of human CC cells. Human CC cells were seeded in 12-well plates containing climbing sheets for 24 h. The experimental group was exposed to 5mM butyrate for 48 h and then washed three times with PBS. Treated cells were fixed with 4% paraformaldehyde for 30 min, resuspended in PBS with 0.3% Triton X-100 and incubated at room temperature for 5 min. TUNEL solution (50µL) was added to the sample, followed by incubation at 37 °C for 60 min in the dark. Antifading mounting medium (with DAPI, Solarbio Science & Technology Co., LTD. Beijing, China) was used for sealing and the stained CC cells were examined with a fluorescence microscope (Becton Dickinson, FACSCantoII).
Tunel apoptosis assay kit
Manufactured by Yeasen
Sourced in China
The TUNEL apoptosis assay kit is a laboratory tool designed to detect and quantify programmed cell death, or apoptosis, in various cell types. The kit utilizes the TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) method, which labels the fragmented DNA that is characteristic of apoptotic cells. This technique allows for the identification and analysis of cells undergoing apoptosis.
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6 protocols using tunel apoptosis assay kit
1
Apoptosis Analysis of Human Colorectal Cancer Cells
2
Evaluation of Apoptosis in Liver Tissue
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Liver tissues of the IR+sevoflurane, the IR and the control groups were collected, fixed using 4% paraformaldehyde for 24 h at 25°C and 5 µm frozen sections were prepared. TUNEL immunohistochemistry analysis was performed using the TUNEL Apoptosis Assay kit (Shanghai Yeasen Biotech Co., Ltd., Shanghai, China). First, tissue slides were deparaffinized twice for 10 min at room temperature using xylene and treated with proteinase K (20 µg/ml; Sigma-Aldrich; Merck KGaA) for 20 min at 25°C. Tissue sections were equilibrated in the kit's equilibration buffer (100 µl) for 5 min at 25°C. FITC-12-dUTP Labeling mix containing recombinant terminal deoxynucleotidyl transferase (100 µl) was added to the sections and samples were incubated at 37°C in a humidity chamber (plastic box with PBS) for 1 h. Sections were incubated with DAPI at 25°C for 5 min. Samples were imaged using a fluorescence microscope (magnification, ×400) and five random fields were selected for each sample.
3
Quantifying Apoptosis Using TUNEL Assay
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The cell apoptosis rate was also determined using a TUNEL apoptosis assay kit (Yeasen Biotechnology, Shanghai, China). After 48 h of incubation, the transferred cells in 6-well plates were fixed with 4% paraformaldehyde for 30 min. Next, the cells were blocked with 3% H2O2 for 10 min in the dark after washing three times with PBS. Afterward, the cells were washed and incubated with 0.2% TritonX-100 (100 μL) to increase cell permeability for 5 min at room temperature. Thereafter, 50 μL of TUNEL reaction solution was added to the sample, which was then incubated for 1 h at 37°C in the dark. Finally, the sample was stained with DAPI, which was applied to visualize the cell nuclei, and analyzed using a fluorescent microscope (Nikon, Japan) [22 (link)].
4
Histological and Immunohistochemical Analysis of Intervertebral Disc Degeneration
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The harvested intervertebral disc specimens were fixed with 4% paraformaldehyde at room temperature for 48 h and decalcified with 10% ethylenediaminetetraacetic acid (EDTA; pH 7.4) at 37 °C for 30 days. Then, the specimens were embedded in paraffin blocks and sectioned at a thickness of 5 μm along the midsagittal orientation. For hematoxylin and eosin (HE) and safranin O-fast green (S-O) staining, all procedures were performed according to the manufacturer’s guidance (Solarbio, Beijing, China). Histological scores were determined as previously described (<5, normal intervertebral discs; 6–11, moderate IDD; and 12–14, severe IDD)33 (link).
For immunohistochemical experiments, the sliced sections were incubated with primary antibodies against O-GlcNAc (diluted 1:100), FAM134B (diluted 1:150) and p16 (diluted 1:150) at 4 °C overnight. After washing three times with PBS, the sections were incubated with the appropriate secondary antibodies at room temperature for 50 min and counterstained with hematoxylin. Then, the sections were imaged and analyzed with a digital slide scanner (S360, Hamamatsu, Japan). Furthermore, apoptosis was assessed using a TUNEL apoptosis assay kit (Yeasen) according to the manufacturer’s instructions, and images were acquired with a fluorescence microscope (Olympus IX71).
For immunohistochemical experiments, the sliced sections were incubated with primary antibodies against O-GlcNAc (diluted 1:100), FAM134B (diluted 1:150) and p16 (diluted 1:150) at 4 °C overnight. After washing three times with PBS, the sections were incubated with the appropriate secondary antibodies at room temperature for 50 min and counterstained with hematoxylin. Then, the sections were imaged and analyzed with a digital slide scanner (S360, Hamamatsu, Japan). Furthermore, apoptosis was assessed using a TUNEL apoptosis assay kit (Yeasen) according to the manufacturer’s instructions, and images were acquired with a fluorescence microscope (Olympus IX71).
5
Apoptosis Detection in Cells and Cartilage
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The cells and cartilage tissues were detected by TUNEL, respectively. TUNEL Apoptosis Assay Kit (40306ES50, Yeasen Biotechnology) was applied to detect the apoptosis rate. The cleaned slipper was fixed with paraformaldehyde for 30 min and cleaned again. The Proteinase K working solution was treated for 20 min. Subsequently, the slides were dripped with the Equilibration Buffer until soaked and incubated for 30 min. After absorbing part of the liquid, TdT incubation buffer solution and DAPI working solution were successively added. Before adding DAPI working solution, tissue-derived sections needed to be reacted with Streptavidin-TRITC labeled working solution for 30min at 37 °C in the dark. This procedure is not required for cell-derived sections. The slippers were detected under a fluorescence microscope after being shielded from light.
6
Detecting Apoptosis in Renal Tissues
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Apoptotic cells in renal tissues were detected with a TUNEL Apoptosis Assay Kit (YEASEN) according to the manufacturer's instructions. The sections were deparaffinized and rehydrated through xylene/ethanol, then treated with proteinase K (Beyotime) for 30 min at 37°C. Afterwards, the slides were incubated with TUNEL detection solution for 1 h. The cell nuclei were stained with DAPI and exhibited the blue light. The images were captured under a fluorescence microscope (Olympus).
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