For the apoptosis assay, A549 and H1299 cells were transfected and cultured as stated. Cell apoptosis was assayed by using an enzyme-linked immunosorbent assay (ELISA) for DNA fragments (Cell Death Detection ELISA assay, Roche, Mannheim, Germany) according to the methods described previously [23 (link)].
Cell death detection elisa assay
Manufactured by Roche
Sourced in Switzerland
The Cell Death Detection ELISA assay is a quantitative sandwich-enzyme-immunoassay for the determination of cytoplasmic histone-associated-DNA-fragments (mono- and oligonucleosomes) after induced cell death. The assay is designed to detect and quantify DNA fragmentation, a hallmark of apoptosis.
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11 protocols using cell death detection elisa assay
1
Cell Apoptosis Assessment by ELISA
2
Detecting Apoptosis in Breast Cancer Cells
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The Cell Death Detection ELISA assay (Roche, Indianapolis, IN, USA) was used to detect apoptosis following the manufacturer's protocol. This assay determines apoptosis by measuring mono- and oligonucleosomes in the lysates of apoptotic cells. Briefly, breast cancer cells MCF7, ZR75-1 and MDA-MB-231 were seeded in 96-well plates and cultured overnight in antibiotic-free media. Cells were then transfected with either 50 nM of miR scrambled control (NC), miR-206 mimic, siRNA-Tbx3 or 100 nM of antagomiR-206 (αmiR-206) using Lipofectamine 2000. Cells were lysed 72 h post transfection. The lysates were placed into a streptavidin-coated microplate and incubated with a mixture of anti-histone-biotin and anti-DNA-peroxidase. The amount of peroxidase retained in the immunocomplex was photometrically determined with ABTS as the substrate. Absorbance was measured at 405 nm. Assays were performed in quadruplicate in three independent experiments.
3
Quantification of Apoptosis by ELISA
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Apoptosis was determined using a Cell Death Detection ELISA assay (Roche Diagnostics, Rotkreuz, Switzerland), based on the detection of cytoplasmic histone-associated DNA fragments in apoptotic cells. Cells were plated in 96-well plates in the appropriate medium (1 × 104 cells per well). After 24 h the cells were serum-deprived for 24 h followed by incubation with 100 µM hydrogen peroxide for 24 h. After fixation of cell lysates, the amount of cell death was quantified using the Cell Death Detection ELISA assay according to the manufacturer´s specifications. Quantification was performed by measuring the absorbance at 405 and 490 nm using a Power Wave 340 ELISA reader (Bio-TEK Instruments, Winooski, USA).
4
Apoptosis Assay with TSA Treatment
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Cells were plated in 6-well plates (50 000 cells/well) and treated or not with TSA during 24h. Apoptosis was quantified using the Cell Death Detection ELISA assay (Roche Molecular Biochemicals), according to the manufacturer's conditions. Values from absorbance measurements at 405 nm were corrected using DNA quantification in separate wells treated in parallel.
5
Quantifying Cell Death Post-Treatment
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Cell lysates equivalent to 1 × 104 per ml after 72 h of treatment with 47Sc-DTPA-cetuximab and/or RUNX3 were collected separately for the assay (Cell Death Detection ELISA assay; Cat #11,544,675,001; Roche, Basel, Switzerland) according to the manufacturer’s instructions. The absorbance at 490 nm was measured. All experiments were performed with triplicate samples.
6
EPO-Mediated Cell Cytoplasmic DNA Assay
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A 96-well plate was coated with AF and seeded with 7.0×103 HRMEC cells/well and attached overnight. The next day, cells were serum deprived and treated with commercial EPO, EPO, or EPO-R76E with or without 2μg/ml sEPOR. After 18 hrs, levels of DNA in the cell cytoplasm were quantified using the Cell Death Detection ELISA assay following the manufacturer’s instructions (Roche, Indianapolis, IN). Absorbance was measured at 409nm, with a 490nm reference. The assay was performed twice with three replicates per assay. A student’s t-test was performed.
7
Apoptosis Induction Assay Protocol
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Apoptosis induction assays were performed after 8 or 24 h incubation with compounds using the Cell Death Detection ELISA assay (Roche). Apoptosis induction is represented relative to DMSO-treated controls (set at 1.0). Error bars denote the SEM.
8
EPO-Mediated Cell Cytoplasmic DNA Assay
9
Caspase-3/7 Activity and DNA Fragmentation Assays
10
Evaluating Anti-VEGF Cytotoxicity in Cells
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After 48 or 72 hrs of treatment with the different doses of anti-VEGF, three colorimetric assays were performed; MTT (Thiazolyl Blue Tetrazolium Bromide) assay (M2128-1G; Sigma-Aldrich, St. Louis, Missouri, USA), lactate dehydrogenase (LDH) assay (11644793001; Roche Life Science, Laval, Quebec, Canada), and the cell death detection ELISA assay (11544675001; Roche Life Science, Laval, Quebec, Canada). The colorimetric assays were performed as per the manufacturers’ instructions. The MTT assay assessed cellular metabolic activity of the experimental cells to the control. The LDH and cell death detection ELISA assays were used to evaluate cytotoxicity of the experimental cells to the control cells by examining cell death through necrosis in the LDH assay and cell death through apoptosis in the ELISA assay. Protein concentrations (23,225; Pierce™ BCA Protein Assay Kit, Thermo Fisher Scientific, Waltham, Massachusetts, USA) were measured for each sample to normalize the absorption data of the LDH and cell death detection ELISA assays.
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