Peritoneal cells collected from WT, KA/KA, and KA/KA;Nos2−/− mice were seeded in six-well culture dishes and washed with PBS following adherence. Remaining peritoneal macrophages were then stimulated with TNFα (10 ng/ml) for 72 h. Following treatment, cells were collected and prepared, according to APO-BrdU TUNEL Assay Kit (Invitrogen, Cat# A23210) instructions and detected using flow cytometry. The percent and mean number of apoptotic cells were recorded as anti-BrdU positive events (per 2.5 × 105 cells). The significance of the data was determined by statistical analysis and Student’s t-test.
Apo brdu tunel assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The APO-BrdU TUNEL Assay Kit is a laboratory tool designed to detect and quantify apoptosis, a form of programmed cell death. The kit utilizes a terminal deoxynucleotidyl transferase (TdT) enzyme to incorporate bromodeoxyuridine (BrdU) into the fragmented DNA of apoptotic cells, which can then be detected using an anti-BrdU antibody.
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Lab products found in correlation
Facscalibur, by BD (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Facs caliber flow cytometer, by BD (3 mentions)
Cellquest software, by BD (3 mentions)
Facscanto 2, by BD (2 mentions)
Fitc conjugated anti annexin 5 antibody and pi, by MBL Life Science (2 mentions)
Accuri c6 flow cytometer, by BD (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Rnase a, by Merck Group (2 mentions)
Click it edu alexa fluor 488 flow cytometry assay kit, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 anti brdu, by Thermo Fisher Scientific (2 mentions)
Live dead fixable for far red dead cell stain kit, by Thermo Fisher Scientific (2 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Epics xl, by Beckman Coulter (1 mentions)
Floid cell imaging station, by Thermo Fisher Scientific (1 mentions)
Facscalibur system, by BD (1 mentions)
Anti β actin monoclonal antibody, by Merck Group (1 mentions)
103 protocols using apo brdu tunel assay kit
1
Peritoneal Macrophage Apoptosis Assay
2
Sperm DNA Fragmentation Analysis by TUNEL
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Terminal deoxynucleotidyl transferase-mediated BrdUTP nick-end labeling (TUNEL) was used to determine sperm DNA fragmentation, following the method described previously.25 (link) In brief, the cells were concentrated by centrifugation, fixed in a solution of ethanol and PBS (70/30, v/v) for 30 min to induce sperm membrane permeabilization, and stored at −20°C until analysis. Cells (approximately 1 × 106) were washed twice with PBS and resuspended in 50 μl of DNA-labeling solution containing 10 μl of reaction buffer, 0.75 μl TdT enzyme, 8.0 μl of BrdUTP (5-Bromo-2’- deoxyuridine 5’- triphosphate) (APO-BrdU™ TUNEL Assay Kit, Invitrogen SA, Barcelona, Spain), and 31.25 μl of dH2O. The cells were incubated in the DNA-labeling solution for 120 min at 37°C. At the end of incubation, 1 ml of rinse buffer was added, and the mixture was centrifuged twice. Finally, the pellet was incubated with anti-BrdU antibody for 30 min at 37°C in a temperature-controlled bath. Negative controls were incubated in the absence of enzyme terminal transferase. The samples were measured by flow cytometry (Epics XL, Beckman Coulter, L’Hospitalet de Llobregat, Barcelona, Spain). Green fluorescence was collected with an FL1 sensor using a 525 nm band-pass filter, determining two populations.
3
Apoptosis Induction in Nasopharyngeal Cancer
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HA, NPC43 and C666-1 cells were incubated with 20 μM C7, 10 μM SAHA or the combination of these drugs for 48 h (for HA cells); with 40 μM C7, 10 μM SAHA or the combination of these drugs for 72 h (for NPC43 and C666-1 cells). Following incubation, both floating and adherent cells were collected and washed twice with PBS. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was then conducted with APO-BrdU TUNEL Assay Kit (Invitrogen, Waltham, MA, USA) following manufacturer’s instructions. The stained cells were detected by flow cytometry (LSRII; BD Biosciences) and data were analyzed by FlowJo software (Tree Star, Ashland, OR, USA).
4
Ginsenoside Rg1 Induces DNA Damage
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After treatment with different concentrations (0–10 μM) of ginsenoside Rg1 for 24 h, the cells were harvested and washed with PBS. DNA strand breaks in MDA-MB-231 cells were detected by the APO-BrdU TUNEL assay kit (Invitrogen) as per the manufacturer's instructions. Samples were analyzed with fluorescent microscopy (Floid Cell Imaging Station, Invitrogen).
5
Flow-based DNA Cleavage Monitoring
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Flow‐based monitoring of DNA cleavage was accomplished using the APO‐BrdU TUNEL Assay kit (Invitrogen). Fixed cells were washed and prepared for DNA nicking/propidium iodide labeling according to manufactures instructions. Cells analyzed using a FACSCalibur system (Becton Dickinson, San Jose, CA), and data analyzed using FlowJo (v8.7).
6
Evaluation of Parasite DNA Fragmentation
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Fragmentation of parasite DNA after exposure with the test compound was evaluated by TUNEL assay using the APO-BrdU TUNEL Assay Kit (Invitrogen) for the detection of DNA fragmentation caused by the effect of drug treatment, and utilizes 5-Bromo-2′-Deoxyuridine 5′-Triphosphate (BrdUTP) and Terminal deoxynucleotidyl transferase (TdT) enzyme. This was carried out following the manufacturer’s protocol, and exactly as previously described16 (link). The treated samples were analysed by flow cytometry using both FL3- width and Anti-BrdU FITC detectors and cell Quest software. The data obtained was analysed with FlowJo 10 software.
7
Biocompatible Polymer-based Anticancer Therapy
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Polycaprolactone diol (MW 2000), poly(ethylene glycol) diacid (MW 600), thiazolyl blue tetrazolium bromide (MTT), carmustine, curcumin, Monoclonal anti β actin antibody (A5316) were used as received from Sigma-Aldrich. Live dead assay kit and Apo-BrdU TUNEL Assay kit was purchased from Invitrogen. Cleaved Caspase 3 and cleaved PARP Antibody (Apoptosis sampler kit Cell signaling technologies #9664). DeadEnd Colorimetry TUNEL system (G7130) was from Promega. Cell culture inserts were purchased from Millipore (Cat No. MCSP24H48) and Hi media (TCP078).
Sprague Dawley rats 6–8 weeks old were used for material biocompatibility study and NOD-SCID mice 4–6 weeks of age (18–20 g) were used for xenograft tumor study. Animals were maintained in Animal Research facility, Rajiv Gandhi Centre for Biotechnology (RGCB) and housed in cages with food and water ad libitum. Animal experiments were carried out in full accordance with the rules and regulations of the Government of India’s Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). The Institutional Animal Ethics Committee (IAEC) of Rajiv Gandhi Centre for Biotechnology, gave their approval to all animal experiments under the protocol no IAEC/732/GSV/2019 for brain biocompatibility and IAEC/750/GSV/2019 for NOD-SCID mice xenograft-based drug delivery system efficacy study.
Sprague Dawley rats 6–8 weeks old were used for material biocompatibility study and NOD-SCID mice 4–6 weeks of age (18–20 g) were used for xenograft tumor study. Animals were maintained in Animal Research facility, Rajiv Gandhi Centre for Biotechnology (RGCB) and housed in cages with food and water ad libitum. Animal experiments were carried out in full accordance with the rules and regulations of the Government of India’s Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). The Institutional Animal Ethics Committee (IAEC) of Rajiv Gandhi Centre for Biotechnology, gave their approval to all animal experiments under the protocol no IAEC/732/GSV/2019 for brain biocompatibility and IAEC/750/GSV/2019 for NOD-SCID mice xenograft-based drug delivery system efficacy study.
8
Apoptosis Evaluation in Drug-Loaded Hydrogels
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APO-BrdU Tunel assay kit (Invitrogen A23210) was used to analyse DNA fragmentation as a marker of apoptosis caused due to drug released from the hydrogel. Briefly, U251 cells 3×105 were seeded in 6-well plates. Drug-loaded PCL-PEG hydrogel (100 µL) was suspended in cell culture insert above cells with media. Cells were trypsinized and processed after 24, 48, and 72 h post-treatment. Cells were fixed with paraformaldehyde, and DNA double-strand nick was labelled with BrdU DNA labelling solution. Cytotoxicity was analysed by FACS.
9
Quantifying Cellular Apoptosis by TUNEL
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The TUNEL analysis was performed to measure the degree of cellular apoptosis using the APO-BrdU™ TUNEL Assay Kit (Invitrogen), according to the manufacturer’s instructions. Cells were fixed in paraformaldehyde for 15 min. Cells were pre-incubated with 50 µL DNA-labeling solution for 1 h at 37°C and then incubated with 5 µL anti-BrdU-fluorescein isothiocyanate (FITC) antibody for 30 min at room temperature (20°C). Finally, cells were mounted with DakoCytomation fluorescent medium and visualized under a fluorescence microscope (Olympus, Tokyo, Japan). These cells were counterstained with propidium iodide to reveal cell nuclei.
10
ATRA-induced Differentiation Assay
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Cells treated with 5 μM ATRA (7 d) were subjected to APO-BrdU TUNEL Assay kit (Invitrogen) according to the manufacturer’s instructions for immune fluorescence and flow cytometry applications. For analysis of differentiation, cells were labeled with anti-S100 or anti-PMP22 antibodies and Alexa Fluor 488 secondary antibodies. Cells treated similarly but without primary antibody served as internal control. Cells were fixed in 1% ice-cold paraformaldehyde (PFA) and permeabilized in 70% ethanol before labeling with primary antibodies. Labeled cells were analyzed using a BD FACS Canto II. Each flow cytometry analysis was performed on 10.000 cells and experiments were performed 2–3 times.
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