Apoptosis detection was further investigated by the Cell Death ELISAPLUS kit (Roche Molecular Biochemicals, Milan, Italy) and by FITC Annexin V Apoptosis Detection Kit II (BD Pharmigen, USA) following manufacturers’ instructions [43 (link)].
Cell death elisaplus kit
Manufactured by Roche
Sourced in Italy, Germany
The Cell Death ELISAPLUS kit is a laboratory tool designed to quantitatively detect and measure cell death-related analytes in cell culture supernatants and tissue extracts. The kit employs an enzyme-linked immunosorbent assay (ELISA) technique to provide accurate and reliable data on various cell death-related parameters.
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9 protocols using cell death elisaplus kit
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Apoptosis Detection by ELISA and FITC Annexin V
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ZnO-NP-induced Apoptosis Quantification
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ZnO-NP-mediated apoptosis was determined using Cell Death ELISAPLUS kit (Roche, Mannheim, Germany). The method was carried out according to an established protocol [49 (link)].
3
Evaluating Anchorage-Independent Viability
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Anoikis (anchorage-independent viability) assay was performed by culturing cells in suspension on polyhema-coated plates for the indicated amount of time, and cell death was assessed using the Cell Death ELISAPLUS Kit (Roche) according to manufacturer’s instructions. Transwell migration assay was performed as described previously [25 (link)].
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Quantifying NETs in Human Serum
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To quantify NETs in human serum, we employed a capture ELISA based on MPO associated with DNA. For the capture antibody, 5 μg/ml anti-MPO mAb (07-496; Upstate) was coated onto 96-well-plates at 4°C overnight. After washing 3 times (300 μl each), 50 μl of serum samples was added to the wells with 50 μl incubation buffer containing a peroxidase-labeled anti-DNA mAb in Cell Death ELISAPLUS kit (1:25, Roche). The plate was incubated for 4 h, shaking at 300 rpm at room temperature. After 3 washes (300 μl each), 100 μl peroxidase substrate (ABTS) was added to incubate at room temperature in the dark for 20 min. Then absorbance at 405 nm wavelength was measured.
5
Investigating 4MU's Impact on Bladder Cancer
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Bladder cancer and normal urothelial cells (1.5 × 104 cells per well) cultured in growth medium were exposed to 4MU (0–0.6 mM ) either alone or in the presence of HA (50 μg ml−1) or a PI3-K inhibitor LY29400 (0 and 10 μM ) for 48 to 72 h. Following incubation, viable cells were counted (Trypan blue staining). Induction of apoptosis was examined in cells treated with 4MU for 48 h using the Cell Death ELISA Plus Kit (Roche Diagnostics, Indianapolis, IN, USA); results were expressed as apoptosis index (per 5000 cells) (Yates et al, 2015 ; Chanmee et al, 2016 (link)). Matrigel invasion and motility assays were carried out as described previously, except that 4MU and/or HA were added in both chambers of the Transwell. Incubation times for motility and invasion assays were 18 and 48 h, respectively (Lokeshwar et al, 2005b (link), 2006 (link), 2010 (link); Yates et al, 2015 ; Jordan et al, 2016 ). In scratch wound assay, 253J-Lung cells were cultured in 0.1% FBS-containing medium and treated with 4MU.The wound closure was photographed at different time intervals and analysed using the ImageJ Software (https://imagej.nih.gov/ij/ ). Per cent wound closure was calculated as: (A0−Am)/A0 × 100 (where A0 is area at time 0 and Am is area at time of measurement). Data are represented as mean±s.d. (triplicate determination).
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Apoptosis Detection Using ELISA
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Apoptosis detection was further investigated by the Cell Death ELISAPLUS kit (Roche Molecular Biochemicals, Milan, Italy). The test is based on the detection of mono- and oligonucleosomes in the cytoplasmic fraction of cell lysates by biotinylated anti histone-coupled antibodies, and their enrichment in the cytoplasm is calculated as the absorbance of sample cells/absorbance of control cells. The enrichment factor was used as a parameter of apoptosis and shown on the Y-axis as mean ± SE. Experiments were performed according to manufacturer’s instructions.
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BCa Cell Apoptosis Modulation
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BCa and normal urothelial cells (1.5×104 cells/well) cultured in growth medium were exposed to sHA-F (0 – 40-μg/ml) either alone or in the presence of AGF (50-μg/ml) or a PI3-kinase inhibitor LY29400 (0, 10-μM) for 48 to 72 hours. Following incubation, viable cells were counted (Trypan blue staining). For apoptosis assay, cells were treated for 48 hours and apoptosis was measured using the Cell Death ELISA Plus kit (Roche Diagnostics; Indianapolis, as per the manufacturer's instruction; the results were expressed as apoptosis index (per 5,000 cells). Apoptosis index: optical density measurement at 405 nm (reference wavelength 490 nm) and subtraction of the negative control readings.
8
MPO-DNA ELISA Assay Protocol
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MPO-DNA ELISA was adapted from Caudrillier et al. (22 (link)). Briefly, flat bottom 96-well plates (Thermofisher Scientific, MA, USA) were coated with an anti-MPO polyclonal antibody (1:500, #2329755, Millipore, CA, USA) at 4°C overnight. Plates were washed with 0.05% Tween in PBS three times before addition of 20 μL patient serum pre-mixed with anti-DNA peroxidase antibody (1:80, Cell Death ELISA Plus kit, Roche, Germany). Cells were allowed to incubate at room temperature in the dark for 3 h under agitation. Plates were then washed five times prior to addition of the substrate Reserve™ TMB (KPL, PA, USA). The reaction was stopped by adding an equal amount of 1 N HCl; O.D. (450 nm) was measured using a microplate reader (EZ400, Biochrom, UK).
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Quantifying Apoptosis Induced by ABri and Bri1-23
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