Cells (2 × 104/per well) were cultivated in 96-well black plates (Corning®, Corning, NY, USA) with 100 μL of supplemented medium (Sigma®, St. Louis, MO, USA) and incubated for 24 h. Treatments on adherent cells were conducted at 5-20 μg/mL of samples/controls for 24 h. Then, ApoTox-Glo™ Triplex Assay (Promega®, Madison, WI, USA) was used to determine events of viability (proliferation), cytotoxicity (necrosis), and apoptosis (caspase-3/7 activation). Additionally, apoptosis was confirmed by an ApoAlert™ DNA Fragmentation Assay (Clontech®, Kusatsu, Shiga, J.P.). Both assays were performed according to each manufacturer’s instructions. Finally, optical density was measured in a VariosKan® Flash (Thermo-Scientific®, Waltham, MA, USA) [29 (link)].
Apoalert dna fragmentation assay
Manufactured by Takara Bio
Sourced in United States
The ApoAlert™ DNA Fragmentation Assay is a laboratory equipment product offered by Takara Bio. It is designed to detect and quantify DNA fragmentation, a hallmark of apoptosis or programmed cell death. The assay provides a reliable and sensitive method for researchers to analyze and measure the extent of DNA fragmentation in cell samples.
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5 protocols using apoalert dna fragmentation assay
1
Cytotoxicity and Apoptosis Evaluation
2
Apoptosis Detection Methods
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Apoptosis was detected by evaluating DNA fragmentation by TdT-mediated dUTP nick-end labelling (TUNEL) assay (ApoAlert DNA Fragmentation Assay, Clontech Laboratories, Inc., Santa Clara, CA, USA) and agarose gel electrophoresis (Antognelli et al, 2013 (link)).
3
Apoptosis Detection via TUNEL Assay
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Apoptosis was detected by evaluating DNA fragmentation by TdT-mediated dUTP nick-end labeling (TUNEL) assay (ApoAlert® DNA Fragmentation Assay, Clontech Laboratories, Inc., Mountain View, CA, USA) [55] (link).
4
TUNEL Assay for Apoptosis Detection
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Frozen sections 10 μm thick were cut using a cryostat (CM1510 S; Leica Microsystems, Taipei, Taiwan) and air-dried. A TUNEL assay was done using a kit (ApoAlert DNA fragmentation assay; Clontech, Palo Alto, CA) according to the manufacturer's instructions. After the assay, a drop of antifade solution was added, the treated portion of the slide was covered with a coverslip, and the edges were sealed with clear nail polish. Slides were viewed within 2 h under a fluorescence microscope (BX 51; Olympus Taiwan, Taipei, Taiwan) equipped with a 40x objective and a dual filter set for green fluorescence (488 nm) and blue fluorescence (461 nm). Images were collected using the microscope fitted with an Olympus America camera and AnalySIS (Soft Imaging Systems, Münster, Germany).
5
Nuclear DNA Fragmentation Assay
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The nuclear DNA fragmentation was evaluated by a commercial kit (ApoAlert® DNA fragmentation Assay, Clontech Laboratories, Inc., Mountain View, CA, USA) in accordance with the manufacturer’s instructions, as previously reported [54 (link)].
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