Apoalert dna fragmentation assay kit

Manufactured by BD

Sourced in United States

The ApoAlert™ DNA Fragmentation Assay kit is a laboratory product designed to detect and quantify DNA fragmentation, a hallmark of apoptosis or programmed cell death. The kit provides reagents and protocols for the analysis of DNA fragmentation using a fluorometric method.

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Lab products found in correlation

Facscan flow cytometer, by BD (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Spectramax m5, by Molecular Devices (1 mentions) Cellquest version 5, by BD (1 mentions) Lipid peroxidation assay kit, by Abcam (1 mentions) Eclipse ti, by Nikon (1 mentions) Laser confocal microscope, by Zeiss (1 mentions) Lab tek chamber slide, by Thermo Fisher Scientific (1 mentions) P erk1 2, by Cell Signaling Technology (1 mentions) Phosphotyrosine705 stat3, by Cell Signaling Technology (1 mentions) Gapdh hrp, by Santa Cruz Biotechnology (1 mentions) Pd98059, by Merck Group (1 mentions) Stat3, by Cell Signaling Technology (1 mentions) Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions) Bcl 2, by Cell Signaling Technology (1 mentions) Erk1 2, by Cell Signaling Technology (1 mentions) Rapamycin sirolimus, by LC Laboratories (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions) Cell counting kit 8 solution, by Dojindo Laboratories (1 mentions) Ham s f12 medium, by Thermo Fisher Scientific (1 mentions)

12 protocols using apoalert dna fragmentation assay kit

Apoptosis Quantification in Vascular Smooth Muscle Cells

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VSMC lysates were prepared and measured using a caspase-3 ELISA Kit (no. KGA203, Nanjing KeyGEN BioTECH, Co., Ltd., Nanjing China). In brief, 50 µl supernatant was mixed with 2X reaction buffer (50 µl) and dithiothreitol (0.5 µl). Immunocomplexes were incubated with 5 µl peptide substrate in assay buffer for 2 h at 37°C. Release of p-nitroaniline was measured at 405 nm using an ELISA reader (SpectraMax M5; Molecular Devices, LLC., Sunnyvale, CA, USA) according to the manufacturer's instructions.
Quantitative assessment of apoptotic cells was performed using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method, examining DNA-strand breaks during apoptosis with the ApoAlert DNA Fragmentation Assay kit (BD Biosciences, Franklin Lakes, NJ, USA). Briefly, cells were incubated at 37°C in hypoxic conditions for 48 h. Cells were trypsinized, fixed with 4% paraformaldehyde at room temperature for 24 h and permeabilized with 0.1% Triton-X-100 in 0.1% sodium citrate. Cells were washed and incubated with the reaction mixture for 60 min at 37°C, and immediately analyzed using FACScan and the Cellquest program ver. 5.1 (BD Biosciences).

Hao M.X., Wang X, & Jiao K.L. (2017). MicroRNA-17-5p mediates hypoxia-induced autophagy and inhibits apoptosis by targeting signal transducer and activator of transcription 3 in vascular smooth muscle cells. Experimental and Therapeutic Medicine, 13(3), 935-941.

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Quantitative Apoptotic Osteoclast Analysis

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Quantitative assessment of apoptotic osteoclasts (5×10⁴) was performed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) method, which examines DNA-strand breaks during apoptosis, using an ApoAlert™ DNA Fragmentation Assay kit (BD Biosciences, Franklin Lakes, NJ, USA). Cells were trypsinized, fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate. The cells were washed with PBS three times and subsequently incubated with the reaction mixture for 60 min at 37°C. Cells were immediately analyzed using a FACScan flow cytometer equipped with the CellQuest version 5.1 (BD Biosciences).

Ma Y., Yang H, & Huang J. (2017). Icariin ameliorates dexamethasone-induced bone deterioration in an experimental mouse model via activation of microRNA-186 inhibition of cathepsin K. Molecular Medicine Reports, 17(1), 1633-1641.

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Apoptosis and Lipid Peroxidation Assessment in Heart

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Apoptosis was determined by TUNEL staining using an ApoAlert™ DNA Fragmentation Assay kit (BD Biosciences, CA) according to the manufacturer's protocol. Hearts were stored in a 10% formalin solution, and paraffin embedded tissue section was mounted on glass slides. Apoptosis was then assessed in the transverse sections of paraffin sections as previously reported [19 (link)]. Apoptotic cells were examined under a fluorescence microscope (Nikon Eclipse Ti) which were clearly identified with a strong nuclear green fluorescence. All cell nuclei were visualized as blue fluorescence following staining with DAPI. The apoptotic index was expressed as the number of apoptotic cells of all cardiomyocytes per field. Apoptotic rate in the peri-infarct regions was calculated using 6 random fields.
Lipid Peroxidation Assay. Lipid peroxidation in heart was assayed by measuring malondialdehyde (MDA) using a lipid peroxidation assay kit (BioVision, CA, USA) according to the manufacturer's protocol as previously reported [7 (link)].

Filippone S.M., Samidurai A., Roh S.K., Cain C.K., He J., Salloum F.N., Kukreja R.C, & Das A. (2017). Reperfusion Therapy with Rapamycin Attenuates Myocardial Infarction through Activation of AKT and ERK. Oxidative Medicine and Cellular Longevity, 2017, 4619720.

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TUNEL Assay for Apoptosis Detection

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The Apo Alert DNA fragmentation assay kit (BD Biosciences, San Jose, CA, USA) was used for TUNEL assays [40 (link)]. Briefly, cells were seeded in each well of a Lab-Tek chamber slide (Nalge Nunc International, Rochester, NY, USA) at 6 × 10⁴ cells per well, cultured for 72 h to form a monolayer, and treated with IFNγ and TNFα for various durations. The cells were subsequently washed with PBS, fixed using 5% acetic acid in ethanol, washed again with PBS, and treated with 0.2% Triton X-100 for 5 min. After washing, the cells were equilibrated for 10 min at room temperature in 50 μl equilibration buffer supplied with the kit, followed by staining at 37°C for 1 h with 45 μl equilibration buffer, 5 μl nucleotide mix, and 1 μl TdT enzyme, which incorporates FITC-labeled dUTPs onto the 3′-terminal hydroxyl group of DNA fragments. Nuclei were then counterstained with propidium iodide (PI), and TUNEL-stained (apoptotic) cells were enumerated using a confocal laser microscope (Carl Zeiss, Oberkochen, Germany) at an excitation wavelength of 494 nm and an emission wavelength of 518 nm. The number of apoptotic cells was normalized to the total number of PI-stained cells.

Iguchi M., Hiroi M., Kanegae H, & Ohmori Y. (2018). Costimulation of Murine Osteoblasts with Interferon-γ and Tumor Necrosis Factor-α Induces Apoptosis through Downregulation of Bcl-2 and Release of Cytochrome c from Mitochondria. Mediators of Inflammation, 2018, 3979606.

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Apoptosis Pathway Regulation Protocol

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Rapamycin (Sirolimus®) was purchased from LC Laboratories (MA). PD98059, an ERK inhibitor, was purchased from Sigma Aldrich (St. Louis, MO). ApoAlert™ DNA Fragmentation Assay kit was purchased from BD Biosciences, Palo Alto, CA. DAPI was bought from Vector Laboratories, Inc. CA. Antibodies for phospho-serine⁴⁷³ AKT, AKT, p-S6, S6, p-ERK1/2, ERK1/2, p-P38, P38, phospho-tyrosine⁷⁰⁵-STAT3, STAT3, Bcl-2, and Bax were purchased from Cell Signaling Technology. SOD-2, ferritin heavy chain, and GAPDH-HRP were purchased from Santa Cruz Biotechnology.

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Cell Proliferation and Apoptosis Assay

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Example 3

ELA cells were cultured as monolayers. Positive and negative cultures were set up in parallel. At days three and five, cultures were rinsed with PBS, detached with trypsin-EDTA, centrifuged, and resuspended in media. Duplicate aliquots were placed into 96-well plates, and 10 μl of Cell Counting Kit-8 solution (Dojindo Molecular Technologies Inc., Gaithersburg, Md.) was added to each well. Following a three hour incubation at 37° C., A450 was measured using a Victor5 Light Luminescence Counter (PerkinElmer Life Sciences, Boston, Mass.) and compared with standards of known cell numbers. To detect apoptotic cells, cultures were fixed and stained with the fluorescence-based ApoAlert DNA Fragmentation Assay Kit (BD Biosciences) following the manufacturer's protocol.

US11535825B2. Adult stem cell compositions and methods of identification and isolation (2022-12-27). TACS Bio, Inc. [US]. Inventors: Keith D. Crawford [US], Baldev Vasir [US], John Garvey [US].

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Investigating Gastric Adenocarcinoma Cell Viability

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The AGS cell line (Dainippon Pharmaceutical Co. Ltd., Osaka, Japan) was originally cultured from stomach adenocarcinoma cells obtained prior to any anti-cancer treatment (Barranco et al., 1983) . Sodium acetate was purchased from Sigma-Aldrich (St. Louis, MO, USA). The Quick Cell Proliferation Assay and Annexin V-PE (phycoerythrin) Apoptosis Detection kits were purchased from Medical & Biological Laboratories (Aichi, Japan). The lactate dehydrogenase (LDH)-cytotoxicity assay kit was purchased from Biovision (Milpitas, CA, USA) and the ApoAlert DNA Fragmentation Assay kit was purchased from BD Biosciences (Franklin Lakes, CA, USA). Caspase-3, -6, -8, and -9 colorimetric protease assays were purchased from Beyotime (Shanghai, China). The DNA Fragmentation Assay kit was purchased from BDBiosciences. Ham's F12 medium and fetal bovine serum were purchased from Gibco (Thermo Fisher Scientific, Waltham, MA, USA).

Xia Y., Zhang X.L., Jin F., Wang Q.X., Xiao R., Hao Z.H., Gui Q.D, & Sun J. (2016). Apoptotic effect of sodium acetate on a human gastric adenocarcinoma epithelial cell line. Genetics and molecular research : GMR, 15(4).

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Matrine Induces Apoptosis in Cells

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The cells (1×106) were treated with 0, 0.2, 0.5, 0.8 and 1.0 mg/ml matrine for 48 h at 37°C, and then collected by centrifugation at 5,300 × g for 5 min at 4°C. The pellets were lysed in DNA lysis buffer, containing 10 mM Tris (pH 7.5), 400 mM EDTA and 1% Triton X-100, and then centrifuged at 5,300 × g for 5 min at 4°C. The supernatant obtained was incubated overnight at 37°C with proteinase K (0.1 mg/ml) and then with RNase (0.2 mg/ml) for 2 h at 37°C. Following extraction with phenol chloroform (1:1), DNA was separated on 2% agarose gel and visualized under UV following staining with ethidium bromide. The quantitative assessment of apoptotic cells was assessed using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) method, which was performed to examine DNA-strand breaks during apoptosis using a BD ApoAlert DNA Fragmentation Assay kit (BD Biosciences).

An Q., Han C., Zhou Y., Li F., Li D., Zhang X., Yu Z., Duan Z, & Kan Q. (2016). Matrine induces cell cycle arrest and apoptosis with recovery of the expression of miR-126 in the A549 non-small cell lung cancer cell line. Molecular Medicine Reports, 14(5), 4042-4048.

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Quantitative Apoptosis Assessment by TUNEL

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Quantitative assessment of apoptotic cells was also performed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick endlabeling (TUNEL) method, which examines DNA-strand breaks during apoptosis, using a BD ApoAlert DNA Fragmentation Assay kit (BD Biosciences, Franklin Lakes, NJ, USA). The HSFBs were trypsinized, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton-X-100 in 0.1% sodium citrate. The cells were washed with phosphate buffered saline twice and then incubated with the reaction mixture for 60 min at 37°C. Cells were immediately analyzed using a FACScan flow cytometer and the BD CellQuestTM Pro program (catalog no. 648089, BD Biosciences).

Xiao K., Luo X., Wang X, & Gao Z. (2017). MicroRNA-185 regulates transforming growth factor-β1 and collagen-1 in hypertrophic scar fibroblasts. Molecular Medicine Reports, 15(4), 1489-1496.

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Quantifying Apoptosis in Osteoblasts

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Apoptosis was assessed using Annexin V, a protein that binds to phosphatidylserine (PS) residues which are exposed on the cell surface of apoptotic cells. The osteoblasts were treated with the vehicle (normal glucose; 5.5 mM) or high glucose for 4 days. Following treatment, the cells were washed twice with phosphate-buffered saline (PBS; pH 7.4), and re-suspended in staining buffer containing 1 µg/ml propidium iodide (PI) and 0.025 µg/ml Annexin V-FITC. Double-labeling was performed at room temperature for 10 min in the dark prior to flow cytometric analysis. The osteoblasts were immediately analyzed using FACScan and the CellQuest program. The quantitative assessment of apoptotic cells was also assessed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) method, which examines DNA-strand breaks during apoptosis using a BD ApoAlert™ DNA Fragmentation assay kit. The stained cells were then analyzed using a flow cytometer (FC500; Beckman Coulter, Miami, FL, USA).

WU M., AI W., CHEN L., ZHAO S, & LIU E. (2016). Bradykinin receptors and EphB2/EphrinB2 pathway in response to high glucose-induced osteoblast dysfunction and hyperglycemia-induced bone deterioration in mice. International Journal of Molecular Medicine, 37(3), 565-574.

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