To measure proliferation, 104 o-GSCs were seeded, and the total cell number counted 3 days later. To measure apoptosis, 105 o-GSCs were plated into 24-well plates coated with poly-d -lysine (50 μg ml−1) and fibronectin (10 μg ml−1) in complete NSC medium. After 3 days, cells were grown in NSC medium without N2, B27, FGF, and EGF for 48 h, fixed in 4% paraformaldehyde (PFA), and apoptosis quantitated using a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) kit (Sigma-Aldrich). MK2206 (Fisher Scientific, S1078) was purchased from Fisher Scientific.
Tunel kit
Manufactured by Merck Group
Sourced in United States, Germany
The TUNEL kit is a laboratory tool used to detect and quantify DNA fragmentation, a hallmark of apoptosis or programmed cell death. The kit provides a method for the in situ labeling of DNA strand breaks, allowing for the identification and analysis of cells undergoing apoptosis.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Hoechst 33342, by Merck Group (2 mentions)
Dfc700t, by Leica camera (2 mentions)
Cleaved caspase 3, by Proteintech (2 mentions)
β actin, by Proteintech (2 mentions)
Bcl 2, by Proteintech (2 mentions)
Caspase 3, by Cell Signaling Technology (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Fluorescence microscope, by Olympus (2 mentions)
Epifluorescence microscope, by Nikon (1 mentions)
In vitro toxicology assay kit, by Merck Group (1 mentions)
Ecl chemiluminescence detection kit, by Cytiva (1 mentions)
Bca protein assay kit, by Merck Group (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Dcfda cellular ros assay kit, by Abcam (1 mentions)
Sterile 1 pbs, by Thermo Fisher Scientific (1 mentions)
Mitocapture mitochondrial apoptosis detection kit, by Abcam (1 mentions)
Alexa fluor 555 goat anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
63 protocols using tunel kit
1
Measuring Proliferation and Apoptosis in o-GSCs
2
Quantifying Apoptotic Endothelial Progenitor Cells
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Apoptotic EPCs were quantified by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) as previously described [29 (link)]. Briefly, 3 × 106 EPCs were seeded on chamber slides and stained with a TUNEL kit (Sigma-Aldrich), followed by nuclear counter-staining with propidium iodide. A non-TUNEL stained negative control was also performed to rule out non-specific autofluorescence. Stained cells were visualized with a Nikon epifluorescence microscope equipped with a digital camera. Five randomly selected 20X fields were captured, and the total number of positively stained cells per field was calculated as a percentage of the total number of cells per field (stained and unstained).
3
Qishen Yiqi Drop Pill Cytotoxicity Evaluation
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Qishen Yiqi Drop Pill (lot number: 110708) was obtained from Tasly Pharmaceutical Co., Ltd. with 0.5 g per pouch. Dulbecco's modified Eagle's medium (DMEM), foetal bovine serum (FBS) and sterile 1 × PBS were purchased from Gibco (Carlsbad). Primary antibodies including cleaved caspase‐3 polyclonal rabbit antibody, bcl‐2 polyclonal rabbit antibody, bax polyclonal rabbit antibody, p53 monoclonal mouse antibody and β‐actin monoclonal mouse antibody, as well as LY294002, the specific inhibitor of PI3K, were obtained from Abcam. DCFDA Cellular ROS Assay Kit was also purchased from Abcam. Horseradish peroxidase‐conjugated secondary antibodies were obtained from Invitrogen. ECL chemiluminescence detection kit was obtained from Amersham Pharmacia Biotech. RIPA lysis buffer, BCA protein assay kit, TUNEL kit and the in vitro toxicology assay kit based on 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide (MTT) were purchased from Sigma‐Aldrich. Polyvinylidene difluoride (PVDF) membranes were from Bio‐Rad. The MitoCapture mitochondrial apoptosis detection kit was obtained from BioVision. The CytoTox 96® non‐radioactive cytotoxicity assay kit was obtained from Promega. The goat anti‐rabbit Alexa Fluor 555 secondary antibody and 4,6‐Diamidino‐2‐phenylindole (DAPI) were obtained from Thermo Fisher Scientific.
4
Quantifying Cellular Apoptosis via TUNEL Assay
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The apoptosis rate of cells was measured using the TUNEL Kit (Sigma, United States) according to the manufacturer’s instructions. In this procedure, the SH-SY5Y cells were seeded on the coverslip placed on the 24-well plates (BD Falcon, United States) and then treated with MPP+ after being transfected with miRNA mimic (Ribo, China). The coverslips were washed twice with phosphate buffered saline (PBS), and fixed with 4% paraformaldehyde at 4°C for 30 min. After washed three times with PBS, the slices were covered with 50 μL TUNEL reaction mixture solution with deoxynucleotide fluorescein-12-dUTP and incubated at humidified atmosphere at 37°C for 60 min in the dark. After rinsing the slices with PBS for three times, the cells were treated with DAPI solution (Sigma, United States) shading at room temperature for 2 min. Finally, samples were rinsed twice with PBS, and slides were covered with mounting medium containing glycerin reagent (Sinopharm, China) and cover glasses (Sail Brand, China). Since then, the apoptotic cells were observed and photographed with a fluorescence microscope.
5
Cardiac Tissue Analysis Protocol
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After isolation, the hearts were fixed, dehydrated and sectioned. Wheat germ agglutinin (WGA) staining was performed to detect the cardiomyocyte cross-sectional area, and more than 100 cells were counted in each group. Masson staining was used to determine the cardiac fibrotic area. In addition, an anti-p53 antibody (GeneTex), an anti-8-hydroxy-2’-deoxyguanosine (8-OHdg) antibody (Santa Cruz) and an anti-apoptosis-inducing factor (AIF, GeneTex) antibody were used to detect cardiac p53, 8-OHdg and AIF expression, respectively. Finally, apoptotic cardiomyocytes were detected using a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) kit (Sigma).
6
TUNEL Staining of Colon Sections
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For TUNEL staining, colon sections were treated with a TUNEL kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. TUNEL-positive cells were observed under a confocal microscope (FLUOVIEW FV3000, Olympus) and analyzed with Imaris software.
7
Apoptosis Detection in Blastocysts
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Apoptotic cells were detected using a fluorescein isothiocyanateconjugated in situ cell death detection kit (terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]; Promega, Durham, NC, USA). The blastocysts were fixed with 4% paraformaldehyde. The apoptotic cells were stained using a TUNEL kit according to the manufacturer’s instructions, and the nuclei were stained in a solution of 10 mg/mL bisbenzimide (Hoechst 33342, Sigma) for 10 minutes prior to observation with a fluorescence microscope (AX-70; Olympus, Tokyo, Japan). Additionally, the number of cells with TUNEL-positive nuclei was determined using an image capturing system (IMT i-Solution, British Columbia, Canada). The apoptotic index was calculated as the percentage of TUNEL-positive nuclei divided by the total number of nuclei in a single blastocyst.
8
Apoptosis Detection in Myocardial Cells
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To detect apoptotic cells in cultured myocardial cells and tissue, TUNEL staining was performed. The cells and tissue slices were fixed with paraformaldehyde (4%) at room temperature for 1 h. TUNEL kit (Sigma-Aldrich; Merck KGaA) was applied to label apoptotic cells according to the manufacturer's instruction. TUNEL staining was performed at 37°C for 2 h. PBS was used for rinsing. Then, 200 ml DAPI (1 µg/ml, Sigma-Aldrich; Merck KGaA) was applied to label nuclei at room temperature for 20 min. Neutral gum was used to mounting. Images were captured in six fields of view/sample using an inverted fluorescence microscope (Nikon Corporation; scale bar, 100 µm). The apoptotic cells were counted manually.
9
Lung Cancer Cell Line Protocol for In Vitro and In Vivo Studies
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Lewis lung cancer (LLC) cell lines were bought from Kang Lang Biotechnology Co., LTD. (Shanghai, PR China). Dulbecco’s modified eagle medium (DMEM) was purchased from Gibco (Gland Island, NY, United States). The antibody of Caspase-3, Bax, Ki-67, and Bcl-2 were obtained from Abcam Company (Cambridge, United Kingdom). Streptomycin and penicillin were supplied by North China Pharmaceutical Co. LTD., Shijiazhuang, China. 4′,6-diamidino-2-phenyli ndole (DAPI) and 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT) were purchased from Sigma-Aldrich (Shanghai, P. R. China). TUNEL kit was purchased from Sigma-Aldrich (United States). BALB/c mice (5-week-old) weighing 18.0 ± 3.2 g were obtained from the Charles River Laboratories of Beijing. All mouse-related experiments were performed in accordance with the Animal Care and Use guidelines of Jilin University.
10
ESCC Cell Line Culture and Apoptosis Assay
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Human ESCC cell line ECA109, which was purchased from Shanghai Cell Institute of Chinese Academy of Sciences, was cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, United States) supplemented with 10% heat-inactivated fetal bovine serum (Gibco) and 100 μg/mL penicillin-streptomycin. The cells were maintained at 37 °C in a humidified atmosphere of 5% CO2. LCL161 (cat. No. 16169) was purchased from MedChemExpress (MedChemExpress, Monmouth Junction, NJ, United States). The TUNEL kit, thiazolyl blue tetrazolium bromide (MTT), and DMSO were purchased from Sigma (St. Louis, MO, United States). Antibodies against XIAP, Bax, and Caspase-3 were purchased from Cell Signaling Technology.
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