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Cell death detection elisa kit

Manufactured by Merck Group
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The Cell Death Detection ELISA kit is a laboratory tool used to quantitatively measure cell death. It is designed to detect and quantify DNA fragmentation, which is a hallmark of apoptosis or programmed cell death. The kit provides a sensitive, reproducible, and standardized method for the analysis of cell death in various cell types and experimental settings.

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Lab products found in correlation

Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions) Bnip3, by Cell Signaling Technology (1 mentions) P tsc2, by Cell Signaling Technology (1 mentions) Total ulk1, by Cell Signaling Technology (1 mentions) Foetal bovine serum (fbs), by Atlanta Biologicals (1 mentions) Beclin 1, by Cell Signaling Technology (1 mentions) P ulk1, by Cell Signaling Technology (1 mentions) Enhanced chemiluminescence ecl western blot detection system, by PerkinElmer (1 mentions) 1 step ultra tmb elisa substrate solution, by Thermo Fisher Scientific (1 mentions) Synergy 2, by Agilent Technologies (1 mentions) Gw3965, by Merck Group (1 mentions) Simvastatin, by Merck Group (1 mentions) Ku55933, by Merck Group (1 mentions) Sb203580, by R&D Systems (1 mentions) Eht 1864, by R&D Systems (1 mentions) Synergy multi mode reader, by Agilent Technologies (1 mentions) Abts solution, by Merck Group (1 mentions) Chloroquine cq, by MedChemExpress (1 mentions) Versamax microplate reader, by Molecular Devices (1 mentions) Confocal laser scanning microscope, by Zeiss (1 mentions)

Close spelling variants of this product

Cell Death Detection ELISA Cell Death Detection Kit ELISAs

15 protocols using cell death detection elisa kit

1

Autophagy and STAT3 Regulation Mechanisms

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Materials purchased include the following: Foetal bovine serum (Atlanta Biologicals); Enhanced chemiluminescence (ECL) Western blot detection system (Perkin Elmer, Inc.); Protease/Phosphatase Inhibitor Cocktail, cleaved active caspase‐3 (Asp 175); LC3‐I/LC3‐II; SQSTM1/p62, p‐Akt Ser473, HDAC‐6, phospho‐S6 Ribosomal protein Ser235/236, p‐STAT3 Y705, p‐STAT3 Ser727, Acetyl‐STAT3 Lys685, total‐STAT3, p‐AMPKα Thr172, Total‐ΑΜPΚα, p‐ULK1 Ser555, p‐ULK1 Ser638, Total‐ULK1, p‐TSC2 Ser1387, p‐TSC2 Ser1462, Total‐Tuberin/TSC2, Beclin‐1, BNIP3 and Cathepsin‐D antibodies (Cell Signalling Technology, Inc.); Alexa‐Fluor 488 conjugated, Alexa‐Fluor 647, and Cy3 conjugated secondary antibodies (Molecular Probes); Anti‐trimethyl STAT3 Lys180, and Bafilomycin A1, (EMD Biosciences/Millipore Corp.); ULK1 Inhibitor (MRT68921) (MedChemExpress). The ULK1 siRNA and transfection reagent were obtained from Santa Cruz Biotechnology. The cell death detection ELISA kit was purchased from Roche (Millipore Sigma). All chemicals were of the highest purity commercially available.
Bhattacharya S., Yin J., Yang C., Wang Y., Sims M., Pfeffer L.M, & Chaum E. (2022). STAT3 suppresses the AMPKα/ULK1‐dependent induction of autophagy in glioblastoma cells. Journal of Cellular and Molecular Medicine, 26(14), 3873-3890.
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2

Quantification of Plasma Myeloperoxidase-Associated NETs

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Similar to a previous NET ELISA assay [46 (link)], Costar 96-well high binding ELISA plates (Corning, NY, USA) were coated overnight at 4 °C with 5 μg/mL anti-human myeloperoxidase (clone 4A4, Bio-Rad) diluted in 1X coating buffer from the Cell Death Detection ELISA kit (MilliporeSigma, Burlington, MA, USA). Then, plates were washed four times with wash buffer (0.2% Tween-20 in PBS), blocked with 5% non-fat dry milk in PBS for 2 h at room temperature, washed four times, and incubated overnight at 4 °C with plasma samples diluted 1:50 in 1% BSA in PBS. Wells were then washed four times, incubated for 2 h at room temperature with anti-DNA-POD (Cell Death Detection ELISA kit, MilliporeSigma) diluted 1X in 1% BSA in PBS, washed four times, incubated with 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific, Waltham, MA, USA) for 10 min, then stopped with 0.2 N H2SO4. Absorbance was read at 450 nm with 540 nm plate correction, using a Synergy 2 plate reader (BioTek) and normalized to a neutrophil NET standard. The neutrophil NET standard was generated by incubating a known number of purified neutrophils with phorbol 12-myristate 13-acetate and ionomycin overnight at 37 °C with 5% CO2. After 100% NETosis was visualized, NETs were scraped from the plate and stored at −80 °C.
Mergaert A.M., Bawadekar M., Nguyen T.Q., Massarenti L., Holmes C.L., Rebernick R., Schrodi S.J, & Shelef M.A. (2019). Reduced Anti-Histone Antibodies and Increased Risk of Rheumatoid Arthritis Associated with a Single Nucleotide Polymorphism in PADI4 in North Americans. International Journal of Molecular Sciences, 20(12), 3093.
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3

NET Quantification from PRP-Stimulated Neutrophils

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A total of 1.5 × 106 neutrophils were cultured in RPMI culture medium for 3.5 h in the presence of PRP, as described in the above section. Unstimulated neutrophils served as control (control NETs). Afterwards, the medium was removed, and cells were washed twice with pre-warmed RPMI medium. NET structures were collected in the supernatant phase, upon vigorous agitation of the culture plate and centrifugation at 20 ×g for 5 min [31 (link),32 (link)]. They were kept undigested at -20 °C till analyzed. The quantification of NETs in supernatants was assessed by MPO/DNA complex ELISA (Cell Death Detection ELISA Kit, Merck), as previously described [33 (link)].
Chrysanthopoulou A., Antoniadou C., Natsi A.M., Gavriilidis E., Papadopoulos V., Xingi E., Didaskalou S., Mikroulis D., Tsironidou V., Kambas K., Koffa M., Skendros P, & Ritis K. (2023). Down-regulation of KLF2 in lung fibroblasts is linked with COVID-19 immunofibrosis and restored by combined inhibition of NETs, JAK-1/2 and IL-6 signaling. Clinical Immunology (Orlando, Fla.), 247, 109240.
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4

Quantifying NET-specific MPO-DNA Complexes

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NET-specific MPO-DNA complexes were measured in (a) EDTA plasma or (b) in vitro–generated NET structures. The method was conducted in accordance to the manufacturer’s instructions (Cell Death Detection ELISA Kit, 11544675001, Merck, Kenilworth) and as previously described (57 (link)).
Chrysanthopoulou A., Gkaliagkousi E., Lazaridis A., Arelaki S., Pateinakis P., Ntinopoulou M., Mitsios A., Antoniadou C., Argyriou C., Georgiadis G.S., Papadopoulos V., Giatromanolaki A., Ritis K, & Skendros P. (2021). Angiotensin II triggers release of neutrophil extracellular traps, linking thromboinflammation with essential hypertension. JCI Insight, 6(18), e148668.
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5

Lipid Starvation and Rescue in Mouse Hepatocytes

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Mouse primary hepatocytes were isolated following a 2-step collagenase digestion protocol and then cultured in William’s E medium with 10% FBS on collagen-coated plates. For lipid starvation, cells were rinsed with serum-free medium and then placed in medium containing 10% delipidated FBS (Gemini Bio). We prepared 30% lipid medium by mixing normal FBS and delipidated FBS at a ratio of 3:7. Cell death was evaluated using the Cell Death Detection ELISA Kit (Merck). GW3965 (Sigma-Aldrich) was dissolved in DMSO and added to medium at 5 μM. C20:4 (Tokyo Chemical Industry Co.) was dissolved in ethanol. For experiments using adenoviral vectors, mouse primary hepatocytes were infected with each adenoviral vector at 24 hours after isolation. After overnight incubation, the virus-containing medium was removed and replaced with fresh medium. Cells were infected with Ad-Cre at a multiplicity of infection (MOI) of 60 for Cre-loxP–dependent gene recombination. PC(16:0_20:4) and PC(18:0_20:4) were purchased from Avanti Polar Lipids, and ER-targeting liposome (PC[16:0_20:4]:PC[18:0_20:4]:dioleoyl-phosphatidylethanolamine:dipalmitoyl-phosphatidylserine at a molar ratio of 1:1:2:1) was generated by Beacle Inc.
Kawamura S., Matsushita Y., Kurosaki S., Tange M., Fujiwara N., Hayata Y., Hayakawa Y., Suzuki N., Hata M., Tsuboi M., Kishikawa T., Kinoshita H., Nakatsuka T., Sato M., Kudo Y., Hoshida Y., Umemura A., Eguchi A., Ikenoue T., Hirata Y., Uesugi M., Tateishi R., Tateishi K., Fujishiro M., Koike K, & Nakagawa H. (2022). Inhibiting SCAP/SREBP exacerbates liver injury and carcinogenesis in murine nonalcoholic steatohepatitis. The Journal of Clinical Investigation, 132(11), e151895.
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6

Evaluating Apoptosis Rates via ELISA

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Based on the DNA fragmentation process of cells in apoptosis can be represented by oligomer generation, we evaluated the apoptosis rate using a Cell Death Detection ELISA kit (Merck, Darmstadt, Germany). All procedures of the operation proceeded according to its manufacturers’ protocol.
Guo X., Li Y., Wan B., Lv Y., Wang X., Liu G, & Wang P. (2023). ETV1 inhibition depressed M2 polarization of tumor-associated macrophage and cell process in gastrointestinal stromal tumor via down-regulating PDE3A. Journal of Clinical Biochemistry and Nutrition, 72(2), 139-146.
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7

Quantitative Analysis of Cellular Signaling

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Antibodies directed against total (Catalog# 2524) and phosphorylated (Serine-15) p53 (Catalog# 9284) were from Cell Signaling Technology (Danvers, MA). Antisera against total (Catalog# ab78) and phosphorylated (Serine-1981) ATM kinase (Catalog# ab36810) were obtained from Abcam (Cambridge, MA). Published evidence from several laboratories have confirmed the specificity of antibodies utilized in this study [30 (link), 33 (link)]. IRDye® 800CW anti-rabbit and anti-mouse were obtained from LICOR (Lincoln, NE). KU-55933, Simvastatin and Cell Death Detection ELISA® kit were from Sigma (St. Louis, MO). EHT1864 and SB203580 were from R&D Systems (Minneapolis, MN). GGTI-2147 was purchased from VDM Biochemicals (Bedford, OH). NE-PER® Nuclear and Cytoplasmic Extraction Reagents were from Thermo Scientific (Waltham, MA).
Sidarala V, & Kowluru A. (2017). Exposure to chronic hyperglycemic conditions results in Ras-related C3 botulinum toxin substrate 1 (Rac1)-mediated activation of p53 and ATM kinase in pancreatic β-cells. Apoptosis : an international journal on programmed cell death, 22(5), 597-607.
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8

Quantifying NET Release and MPO-DNA Complexes

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NETs release in culture and plasma was quantified with Quanti-IT Pico
Green double stranded (ds) DNA kits (Thermo Fisher Scientific) using lambda DNA
as quantitation standards in accordance with manufacturers recommendations. To
capture NET fragments from BALF 100 μg of rabbit polyclonal Chip grade
Abs specific for citrullinated histone H3 (Abcam) or 500 μg control
rabbit polyclonal Abs (Jackson Labs) were conjugated to 1.7 mg of 0.3 mm
diameter IDC UltraClean Amidine Latex beads (Fisher Thermo Scientific) in
accordance with manufacturers recommendations. BALF was split into two 1.5 ml
aliquots and incubated with either 250 μg H3 citrullinated Ab-beads or
control Ab-beads at 4 °C for 3 hours. Beads were then washed twice in
cold PBS and directly quantified for dsDNA content with Pico Green relative to
dsDNA content adsorbed to control Ab-beads. MPO-DNA complex detection in
circulating plasma was conducted with MPO ELISA kit plates (ThermoFisher
Scientific) precoated with MPO capture Abs. Then following plasma incubation for
3 hours at 4° C and 3 washes of cold ELISA washer buffer plates were
incubated with anti-DNA peroxidase conjugated Abs from a Cell Death Detection
ELISA kit (Sigma), washed three times, and incubated with ABTS solution (Sigma)
for 20 minutes. Complexes were quantified as optical density at 405 nm using a
Synergy Multimode reader (Biotek).
Scozzi D., Wang X., Liao F., Liu Z., Zhu J., Pugh K., Ibrahim M., Hsiao H.M., Miller M.J., Yizhan G., Mohanakumar T., Krupnick A.S., Kreisel D, & Gelman A.E. (2018). Neutrophil extracellular trap fragments stimulate innate immune responses that prevent lung transplant tolerance. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 19(4), 1011-1023.
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9

Evaluating Cytotoxicity of SBI-0206965 and Chloroquine

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A498 and ACHN cells (1 × 105/well) were evenly distributed and grown in 100-mm culture dishes overnight and then treated with Earle's Balanced Salt Solution (EBSS) and SBI-0206965 (Cat. No. HY-16966, MedChem Express, Shanghai, China) at various concentrations (0, 5, 10, and 20 μg/mL) or chloroquine (CQ; Cat. No. C6628; Sigma-Aldrich) for 24 h. Cells were harvested and assayed using a Cell Death Detection ELISA Kit (Cat. No. 11544675001, Roche, Switzerland) following the manufacturer's instructions.
Lu J., Zhu L., Zheng L.P., Cui Q., Zhu H.H., Zhao H., Shen Z.J., Dong H.Y., Chen S.S., Wu W.Z, & Tan J.M. (2018). Overexpression of ULK1 Represents a Potential Diagnostic Marker for Clear Cell Renal Carcinoma and the Antitumor Effects of SBI-0206965. EBioMedicine, 34, 85-93.
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10

Quantifying NET Levels in Serum

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Circulating DNA levels have been reported as a marker of NET formation19 (link). We used a capture enzyme-linked immunosorbent assay (ELISA) to quantify the NET levels in the serum as previously described14 (link). The commercially available Cell Death Detection ELISA kit (Sigma, 11544675001) and the MPO Mouse ELISA kit (Hycult Biotech, Frontstraat 2a, 5405 PB Uden, The Netherlands) were used to determine the NET concentrations in the serum. The optical density (OD) between 405 and 490 nm was measured to reflect the concentration of cfDNA in the serum. Values for the formation of soluble NETs are presented as the fold induction with the values of the sham group set to 1.
Wang S., Xie T., Sun S., Wang K., Liu B., Wu X, & Ding W. (2018). DNase-1 Treatment Exerts Protective Effects in a Rat Model of Intestinal Ischemia-Reperfusion Injury. Scientific Reports, 8, 17788.
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