Cell death induced by ethanol and cigarette smoke extract in AR42J cells and primary mouse and human acini was measured by propidium iodide uptake. Apoptosis and necrosis were further characterized by Annexin-V-FLUOS staining (#11858777001, Roche Molecular Biochemicals) in living cells. Apoptosis was also assessed by measuring internucleosomal DNA fragmentation in cell lysates using Cell Death Detection ELISAPLUS kit (#11774425001, Sigma-Aldrich). Detailed information is provided in Supplementary Methods .
Cell death detection elisaplus kit
Manufactured by Merck Group
Sourced in United States, Germany
The Cell Death Detection ELISAPLUS kit is a laboratory equipment product manufactured by Merck Group. It is used for the detection and quantification of DNA fragmentation, which is an indicator of apoptosis or cell death. The kit provides a colorimetric-based enzyme-linked immunosorbent assay (ELISA) method for the measurement of this cellular process.
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19 protocols using cell death detection elisaplus kit
1
Ethanol and Cigarette Smoke-Induced Cell Death
2
Quantifying Apoptotic DNA Fragmentation
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Apoptotic DNA fragmentation is a crucial feature of apoptosis. Therefore, internucleosomal DNA fragmentation was quantitatively assayed by the antibody-mediated capture and detection of cytoplasmic mononucleosome- and oligonucleosome-associated histone‒DNA complexes (Cell Death Detection ELISAPLUS Kit; Sigma-Aldrich). Briefly, SiHaP and SiHaCIS-R cells were cultured in 96-well plates and treated with CIS (30 µM), PTX (4 mM), or PTX + CIS (4 mM + 30 µM) for 24 h. Afterward, the cell culture supernatants were removed. The cells were resuspended in 200 µl of the lysis buffer™ and lysed directly in the wells. Cell lysates were then centrifuged (1,200 rpm, 10 min), and the cytoplasmic fraction (20 µl) was employed to determine DNA fragmentation according to the manufacturer’s standard protocol. Subsequently, absorbance was measured at 405 nm (490-nm filter as a reference wavelength) in a microplate reader. In the DNA fragmentation test, the rate of apoptosis is reflected by the enrichment (fold increase) of mono- and oligonucleosomes accumulated in the cytoplasm. DNA fragmentation was calculated according to the following formula: Rate of Apoptosis = Absorbance of Sample Cells/Absorbance of UCG.
3
Quantifying Apoptosis in Equine ERU
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Serum and VBF samples of horses with healthy eyes and ERU-diseased horses were examined with a Cell Death Detection ELISAPLUS Kit (Sigma 11774425001) in accordance with the manufacturer’s recommendations for evaluating the amount of nucleosomes in those samples.
4
Apoptosis Assay in Adherent Cells
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A total of 24 post-transfection, 1×105 cells were seeded in normal and low-attachment surface 24-well plates. Following incubation for 24 h at 37°C, a Cell Death Detection ELISA PLUS kit (cat. no. 11774425001; Sigma Aldrich; Merck KGaA, Darmstadt, Germany) was used to detect apoptosis, according to the manufacturer's protocol.
5
Quantifying Cell Apoptosis via ELISA
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Cell apoptosis was evaluated by the level of histone-associated DNA fragments (mono- and oligonucleosomes) using the Cell Death Detection ELISAPLUS kit (Sigma-Aldrich, St. Louis, MO, USA), according to the manufacturer’s instructions. Briefly, cell lysates were placed in a streptavidin-coated microplate and incubated with a mixture of anti-histone-biotin and anti-DNA peroxidase. Then, the optical density (405 nm) of the samples was measured on a Spectramax M2 microplate reader (Molecular Devices, San Jose, CA, USA). According to the kit’s manual, 1 × 10−3 OD is equivalent to 1mU of Histone/DNA fragments.
6
Apoptosis Detection via ELISA
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Cell death detection ELISA plus kit (Sigma- Aldrich, USA; #11287100) was used to assess DNA fragmentation as an index of apoptosis using a plate reader (BioTek, USA).
7
Apoptosis Evaluation via ELISA
8
Biomarkers in Arterial Blood Samples
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Blood samples from patients were collected from radial or femoral site of arterial catheter and processed within 1 h. Blood samples were always obtained around midday to limit circadian oscillations of neutrophil phenotypes. Haematological parameters were determined in EDTA-anticoagulated blood on a Cell-Dyn 3700 (Abbott Laboratories). Platelet poor plasma was obtained after two consecutive centrifugations at 1750 g during 15 min at room temperature. Sera were obtained following one centrifugation at 1750 g during 15 min at RT. Plasma and sera were stored at −80 °C until analysis. IL-6 and S100A9 were measured on EDTA-plasma using a Luminex assay kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Nucleosome levels were quantified on EDTA-plasma with the Cell Death Detection ELISA plus kit (Sigma-Aldrich, Overijse, Belgium). Myeloperoxidase quantity and activity were analysed on EDTA-plasma by a method adapted from Franck et al. [11 (link)] using a human myeloperoxidase (MPO) Quantikine ELISA kit (R&D Systems). hs-CRP, CK-MB (Abbott Laboratories, Wavre, Belgium) and high-sensitive troponin T (hs-TnT) (Roche, Machelen, Belgium) were measured in sera by routine immunoassays.
9
Apoptosis Assay in Leiomyoma Cells
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Apoptosis index was determined in parallel experiments using Cell Death Detection ELISA Plus Kit (Sigma-Aldrich). Briefly, cultured leiomyoma cells (1.5 × 103 cells/well) were transfected with either FKBP5 or control siRNAs and treated with steroids. Colorimetric absorbance was measured at 405 nm 48 h after steroid treatment which was 72 h after transfection.
10
Rottlerin-Induced Apoptosis in PaSC
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PaSC were plated in 60-mm dishes and treated with different concentrations of rottlerin. Apoptosis was estimated by measuring internucleosomal DNA fragmentation in media and cell lysates using Cell Death Detection ELISAPLUS kit (#11774425001, Sigma-Aldrich) according to the manufacturer’s recommendation.
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