Human keratinocytes were cultured in 6-well plates and, at 60–80% confluence, treated with DCE, CS and HCS for 1 h, and then stimulated with 50 ng/ml human recombinant TNF-α for 48 h. Apoptosis of keratinocytes was evaluated using the Genzyme TACS Annexin V apoptosis detection kit (R&D Systems) or the Cell Death Detection ELISA Plus kit (Roche Diagnostics). Viable, necrotic and apoptotic cells were analyzed by flow cytometry with a FACScan equipped with Cell Quest software (Becton Dickinson). The amount of nucleosomes was detected and quantified in cell lysates using Cell Death Detection ELISA Plus kit (Roche diagnostics), as per the manufacturer' instructions.
Cell death detection elisaplus kit
Manufactured by Roche
Sourced in Germany, United States, Switzerland, Spain, France, Italy, United Kingdom, China
The Cell Death Detection ELISAPLUS kit is a quantitative in vitro assay used for the detection and quantification of cytoplasmic histone-associated DNA fragments, which are indicative of apoptosis. The kit provides a simple, fast, and reliable method for the measurement of cell death.
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Lab products found in correlation
Brdu cell proliferation assay kit, by Cell Signaling Technology (7 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions)
Human calprotectin elisa kit, by Hycult Biotech (4 mentions)
Quant it picogreen dsdna kit, by Thermo Fisher Scientific (4 mentions)
Multiskan go, by Thermo Fisher Scientific (4 mentions)
Dapi solution, by Roche (3 mentions)
Celltiter blue, by Promega (3 mentions)
Microplate reader, by Bio-Rad (3 mentions)
Caspase glo 3 7 luminescence assay, by Promega (3 mentions)
Human mpo elisa kit, by Abnova (3 mentions)
Apoptotic dna ladder kit, by Roche (3 mentions)
Propidium iodide, by Merck Group (3 mentions)
Fluorescence microscopy, by Zeiss (2 mentions)
Caspase glo 3 7 assay, by Promega (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
Nuclear extract kit, by Active Motif (2 mentions)
Elx800, by Agilent Technologies (2 mentions)
Anti glyceraldehyde 3 phosphate dehydrogenase gapdh antibody, by GeneTex (2 mentions)
312 protocols using cell death detection elisaplus kit
1
Apoptosis Assay for Keratinocytes
2
Quantitative Cytotoxicity Assay for ssRNA40
3
Honokiol and TRAIL-induced Apoptosis
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Caki cells were treated with honokiol alone, TRAIL alone or honokiol plus TRAIL. To measure DNA fragmentation, we used cell death detection ELISA plus kit (Boehringer Mannheim, Indianapolis, IN, USA) according to the manufacturer’s recommendations. The reaction products were analyzed by spectrophotometry (BMG Labtech, Ortenberg, Germany) at 405 and 490 nm (reference wavelength). For DEVDase activity assay, cells were harvested and incubated with reaction buffer containing acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) substrate, as previously described [37 (link)].
4
DAPI Staining and DNA Fragmentation Detection
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For detection of DAPI staining and DNA fragmentation, we used 300 nM DAPI solution (Roche, Mannheim, Germany), and the cell death detection ELISA plus kit (Boehringer Mannheim, Indianapolis, IN, USA) [20 (link)].
5
Apoptosis Detection by DAPI and ELISA
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To identify apoptosis, nuclear morphology was stained with 4′,6′-diamidino-2-phenylindole (DAPI) (Roche, Mannheim, Germany), and detected by fluorescence microscopy. To detect fragmented DNA, we used the cell death detection ELISA plus kit (Boehringer Mannheim, Indianapolis, IN, USA). Cells were lysed, and then centrifuged at 200× g for 10 min. Lysates were used for detection of nucleosomes in cytoplasmic fractions by spectrophotometry.
6
Apoptosis Detection in Kahweol-Sorafenib Treated Cells
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A cell death detection ELISA plus kit (Boehringer Mannheim; Indianapolis, IN) was used to determine the level of apoptosis by detecting fragmented DNA within the nuclei of kahweol-treated cells, sorafenib-treated cells, or cells that were treated with a combination of sorafenib and kahweol. Briefly, each culture plate was centrifuged for 10 min at 200 × g, the supernatant was removed, and the cell pellet was lysed for 30 min. Then, the plate was centrifuged again at 200 × g for 10 min and the supernatant, which contained the cytoplasmic histone-associated DNA fragments, was collected and incubated with an immobilized anti-histone antibody. The reaction products were incubated with a peroxidase substrate for 5 min and were measured by spectrophotometry at 405 and 490 nm (reference wavelength) with a microplate reader. The signals in the wells containing the substrate alone were subtracted as the background.
7
Caspase-mediated cell death analysis
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Caki-1 cells were treated with mdivi-1 alone, cisplain alone or combined treatments. To measure DNA fragmentation, we used cell death detection ELISA plus kit (Boehringer Mannheim, Indianapolis, IN, USA) according to the manufacturer’s recommendations. For caspase activity assay, cells were harvested and incubated with a reaction buffer containing acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA), acetyl-Ile-Glu-Thr-Asp-p-nitroanilide (Ac-IETD-pNA), or Ac-Leu-Glu-His-Asp-p-nitroaniline (Ac-LEHD-pNA) substrate, as previously described [29 (link)].
8
Oxaliplatin and ZFL Induce DAPI Staining and DNA Fragmentation
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For 4′,6′-diamidino-2-phenylindole (DAPI) staining and DNA fragmentation, cells were treated with 25 μM oxaliplatin and/or 2 μM ZFL for 24 h. Caki cells were fixed, washed with PBS, and stained with a 300 nM DAPI solution (Roche, Mannheim, Germany), or DNA fragmentation was detected using a cell death detection ELISA plus kit as described in our study (Boehringer Mannheim, Indianapolis, IN, USA)31 (link).
9
Apoptosis Quantification of Nalm-6 Cells
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Apoptosis of Nalm-6 cells was assayed by the Cell Death Detection Elisa Plus kit (Boehringer Mannheim, Indianapolis, IN, USA) according to manufacturer’s instructions. For this assay, Nalm-6 cells were cultured in FBS-free RPMI-1640 culture medium for 24 h, then collected and subjected to apoptotic cell quantification using monoclonal antibodies directed against DNA and histones. Absorbance was measured at 405 nm.
10
Quantifying Cell Death DNA Fragmentation
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DNA fragmentation was performed using the Cell Death Detection ELISAPLUS kit (Boehringer Mannheim; Indianapolis, USA). Briefly, cells were centrifuged for 10 min at 200 × g, the supernatant was removed, and pellet was lysed for 30 min. After centrifuging the plate again at 200 × g for 10 min, and the supernatant that contained the cytoplasmic histone-associated DNA fragments was collected and incubated with an immobilized anti-histone antibody. The reaction products were incubated with a peroxidase substrate for 5 min and measured by spectrophotometry at 405 nm and 490 nm (reference wavelength) with a microplate reader. The signals in the wells containing the substrate alone were subtracted as background.
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