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GFP-CERTIFIED® Apoptosis/Necrosis detection kit

Multiplex assay that distinguishes between  healthy, early apoptotic, late apoptotic and necrotic cells, compatible with GFP and other green fluorescent probes
ENZ-51002-25 25 assays 157.00 USD
ENZ-51002-100 100 assays 371.00 USD
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  • True multiplexing capabilities with GFP and other green fluorescent probes
  • Readily distinguishes between healthy, early apoptotic, late apoptotic and necrotic cells
  • Optimized for both fluorescence microscopy and flow cytometry applications
  • Suitable for death pathway analysis and drug/toxin studies
The transition from apoptosis to necrosis is a loosely defined continuum that necessitates recognition of the various stages of the process. The display of phosphatidylserine (PS) on the extracellular face of the plasma membrane remains a unifying hallmark of early apoptosis. Phospholipid-binding proteins such as Annexin V bind with a high affinity to PS, in the presence of Ca2+. Given that Annexin V is not cell permeable, the binding of externalized PS is selective for early apoptotic cells. Similarly, loss of plasma membrane integrity, as demonstrated by the ability of a membrane-impermeable DNA intercalating dye to label the nucleus, represents a straightforward approach to demonstrate late stage apoptosis and necrosis. Considering that Annexin V is commonly conjugated to fluorescein (a.k.a. FITC), apoptosis detection in green fluorescent protein (GFP)-expressing cells can be problematic. The spectral overlap between the emission profiles of these two fluorophores makes differentiation between signals difficult or impossible, thus compromising data quality overall. While useful for analysis of apoptosis and necrosis in cells that do not express any fluorescent proteins, the GFP-CERTIFIED® Apoptosis/Necrosis Detection System was specifically designed for use with GFP-expressing cell lines and cells expressing blue or cyan fluorescent proteins (BFPs, CFPs). Additionally, the kit is suitable for use with live or post-fixed cells in conjunction with probes, such as labeled antibodies, or other fluorescent conjugates displaying similar spectral properties as fluorescein or coumarin. This kit includes all the necessary reagents for determination of early and late stages of apoptosis as well as necrosis. An Annexin V-EnzoGold (enhanced Cyanine-3) conjugate enables detection of apoptosis distinct from fluorescein or GFP. The Necrosis Detection Reagent (Red) similar to the red-emitting dye 7-AAD, facilitates late apoptosis and necrosis detection. This kit also includes an Apoptosis Inducer (Staurosporine) for use as a positive control. Detection reagents can be used separately or in combination for either single or multiplexed applications respectively. This kit also provides quick and accurate results via flow cytometry and fluorescence/confocal microscopy.
ENZ-51002 Fig1A-B
FLOW CYTOMETRY. Jurkat cells were mocked-induced with 0.2% DMSO (panel A) or induced with 2uM Staurosporine (panel B) for 4 hrs. at 37°C. After treatment, cells were incubated with a buffer containing Annexin V-Cyanine 3 and the Necrosis Detection Reagent (Red), a far red emitting DNA-intercalating dye, then analyzed by flow cytometry using a 488nm laser with fluorescence detection with FL2 (Apoptosis Detection Reagent) and FL3 (Necrosis Detection Reagent) channels. Mock-induced cells were primarily negative for apoptosis and necrosis. After a 4 hour treatment there were three populations of cells: (1) cells that were viable and not apoptotic or necrotic (Annexin V-Cyanine 3 and Necrosis Detection Reagent negative); (2) cells undergoing apoptosis (Annexin V-cyanine 3 positive and NDR negative); and (3) cells undergoing late-stage apoptosis and early necrosis (Annexin V-Cyanine 3 and Necrosis Detection Reagent positive).
ENZ-51002 IF
GFP-CERTIFIED® Apoptosis/Necrosis Detection Kit (ENZ-51002)
ENZ-51002 Fig2
Excitation (hatched) and emission (solid) spectra for GFP, Annexin V-Cyanine 3 conjugate and Necrosis Detection Reagent (Red). All three dyes are readily excited with a 488nm laser source. The emission maxima of all fluorophores are well separated from one another.
ENZ-51002 Fig3
Jurkat cells stimulated with 2.5 µM Staurosporine for 0-6 h. Flow cytometry results using Jurkat suspension cells, showing early apoptotic (Annexin V-EnzoGold) and late apoptotic/necrosis (Annexin V-EnzoGold and necrosis stain).
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ENZ-51002 Fig1A-B ENZ-51002 IF ENZ-51002 Fig2 ENZ-51002 Fig3

Product Specification

Applications:Flow Cytometry, Fluorescence microscopy, Fluorescent detection
Use/Stability:Reconstituted inducer (staurosporine) should be stored at -20°C.
Handling:Protect from light.
Shipping:Shipped on Blue Ice
Long Term Storage:+4°C
Kit/Set Contains:Apoptosis Detection Reagent (Annexin V-EnzoGold)
Necrosis Detection Reagent
Apoptosis Inducer (Staurosporine)
Binding Buffer (10X)
Technical Info/Product Notes:Application Note:
Image-Based Analysis of a Human Neurosphere Stem Cell Model for the Evaluation of Potential Neurotoxicants

Cited samples:
For an overview on cited samples please click here.

Product Literature References

Cluster microRNAs miR‐194 and miR‐215 suppress the tumorigenicity of intestinal tumor organoids: T. Nakaoka, et al.; Cancer Sci. 108, 678 (2017), Application(s): Flow cytometry using organoid culture of mouse intestinal tumors, Abstract; Full Text
Elaidic Acid, a Trans-Fatty Acid, Enhances the Metastasis of Colorectal Cancer Cells: H. Ohmori, et al.; Pathobiology 84, 144 (2017), Abstract;
Pro-metastatic intracellular signaling of the elaidic trans fatty acid: K. Fujii, et al.; Int. J. Oncol. 50, 85 (2017), Abstract;
Immunotherapy of acute leukemia by chimeric antigen receptor-modified lymphocytes using an improved Sleeping Beauty transposon platform: C.F. Magnani, et al.; Oncotarget 7, 51581 (2016), Application(s): Flow cytometry using KG-1, REH, Nalm-6, and THP-1 cells, Abstract; Full Text
Novel long chain fatty acid derivatives of quercetin-3-O-glucoside reduce cytotoxicity induced by cigarette smoke toxicants in human fetal lung fibroblasts: N. Sumudu, et al.; Eur. J. Pharmacol. 781, 128 (2016), Abstract;
PKLR promotes colorectal cancer liver colonization through induction of glutathione synthesis: A. Nguyen, et al.; J. Clin. Invest. 126, 681 (2016), Application(s): Assessed Apoptosis, Flow cytometry, Abstract; Full Text
Protective effects of β‐casofensin, a bioactive peptide from bovine β‐casein, against indomethacin‐induced intestinal lesions in rats: C. Bessette, et al.; Mol. Nutr. Food Res. 60, 823 (2016), Abstract;
BRCA1, PARP1 and γH2AX in acute myeloid leukemia: Role as biomarkers of response to the PARP inhibitor olaparib: I. Faraoni, et al.; Biochim. Biophys. Acta 1852, 462 (2015), Application(s): Flow cytometry using primary AML cells, Abstract;
Growth-promoting and tumourigenic activity of c-Myc is suppressed by Hhex: V. Marfil, et al.; Oncogene 34, 3011 (2015), Application(s): Flow cytometry using HO15.19 and TGR-1 cells, Abstract;
High throughput proteomic analysis and a comparative review identify the nuclear chaperone, Nucleophosmin among the common set of proteins modulated in Chikungunya virus infection: R. Abraham, et al.; J. Proteomics. 120, 126 (2015), Application(s): Flow cytometry using HEK293 cells, Abstract;
Identification of novel osteogenic compounds by an ex-vivo sp7: luciferase zebrafish scale assay: E. de Vrieze, et al.; Blood 74, 106 (2015), Application(s): Fluorescence microscopy using fish scales, Abstract;
Impaired oxidative phosphorylation regulates necroptosis in human lung epithelial cells: M.J. Koo, et al.; Biochem. Biophys. Res. Commun. 464, 875 (2015), Application(s): Apoptosis/necrosis assay, Abstract;
Influenza virus M2 targets cystic fibrosis transmembrane conductance regulator for lysosomal degradation during viral infection: J.D. Londino, et al.; FASEB J. 29, 2712 (2015), Application(s): Flow cytometry using HEK293 cells, Abstract; Full Text
Inhibition of autophagy exerts anti-colon cancer effects via apoptosis induced by p53 activation and ER stress: K. Sakitani, et al.; BMC Cancer 15, 795 (2015), Application(s): Flow cytometric analysis of apoptosis, Abstract; Full Text
N-terminal functional domain of Gasdermin A3 regulates mitochondrial homeostasis via mitochondrial targeting: P.H. Lin, et al.; J. Biomed. Sci. 22, 44 (2015), Application(s): Fluorescence microscopy using HaCaT and HEK293 cells, Abstract; Full Text
Novel carbocyclic curcumin analog CUR3d modulates genes involved in multiple apoptosis pathways in human hepatocellular carcinoma cells.: K.S. Bhullar, et al. ; Chem. Biol. Interact. 242, 107 (2015), Application(s): Fluorescence microscopy, Abstract;
NPD1-mediated stereoselective regulation of BIRC3 expression through cREL is decisive for neural cell survival: J.M. Calandria, et al.; Cell Death Differ. 22, 1363 (2015), Application(s): Assay, Abstract;
ROS-induced oxidative stress and apoptosis-like event directly affect the cell viability of cryopreserved embryogenic callus in Agapanthus praecox: D. Zhang, et al.; Plant Cell Rep. 34, 1499 (2015), Abstract;
The Adhesion GPCR CD97/ADGRE5 inhibits apoptosis: C.C. Hsiao, et al.; Int. J. Biochem. Cell. Biol. 65, 197 (2015), Application(s): Flow cytometry using HT1080 cells, Abstract;
The effect of plasma-derived activated protein C on leukocyte cell-death and vascular endothelial damage: T. Iba, et al.; Thromb. Res 135, 963 (2015), Application(s): Fluorescence Microscopy using leukocytes, Abstract;
Accumulation of cytosolic calcium induces necroptotic cell death in human neuroblastoma: M. Nomura, et al.; Cancer Res. 74, 1056 (2014), Abstract;
Apoptotic and inhibitory effects on cell proliferation of hepatocellular carcinoma HepG2 cells by methanol leaf extract of Costus speciosus: S.V. Nair, et al.; Biomed. Res. Int. 2014, Article ID 637098 (2014), Application(s): Measurement by flow cytometry, Abstract; Full Text
Combination of antithrombin and recombinant thrombomodulin modulates neutrophil cell-death and decreases circulating DAMPs levels in endotoxemic rats: T. Iba, et al.; Throm. Res. 134, 169 (2014), Application(s): Detection Kit using live cells, Abstract;
Fatty acid esters of ohloridzin induce apoptosis of human liver cancer cells through altered gene expression: S.V. Nair, et al.; PLoS One 9, e107149 (2014), Application(s): Fluorescence microscopy of HepG2 cells, Abstract; Full Text
Flavonoid-enriched apple fraction AF4 induces cell cycle arrest, DNA topoisomerase II inhibition, and apoptosis in human liver cancer HepG2 cells: S. Sudan, et al.; Nutr. Cancer 66, 1237 (2014), Abstract;
Quercetin-3-O-glucoside induces human DNA topoisomerase II inhibition, cell cycle arrest and apoptosis in hepatocellular carcinoma cells: S. Sudan, et al.; Anticancer Res. 34, 1691 (2014), Abstract;
A light-activated NO donor attenuates anchorage independent growth of cancer cells: Important role of a cross talk between NO and other reactive oxygen species: S. Sen, et al.; Arch. Biochem. Biophys. 540, 33 (2013), Application(s): Detection Kit using live cells, Abstract;
Alu Elements in ANRIL Non-Coding RNA at Chromosome 9p21 Modulate Atherogenic Cell Functions through Trans-Regulation of Gene Networks: L.M. Holdt, et al.; PLoS Genet. 9, e1003588 (2013), Application(s): Detection Kit using live cells, Abstract; Full Text
Analysis of protein translocation into the endoplasmic reticulum of human cells: J. Dudek, et al.; Methods Mol. Biol. 1033, 285 (2013), Abstract;
Characterizing virulence-specific perturbations in the mitochondrial function of macrophages infected with Mycobacterium tuberculosis: S. Jamwal, et al.; Sci. Rep. 3, 1328 (2013), Abstract; Full Text
Evaluation of anticancer effects and enhanced doxorubicin cytotoxicity of xanthine derivatives using canine hemangiosarcoma cell lines: T. Motegi, et al.; Res. Vet. Sci. 95, 600 (2013), Application(s): Fluorescence microscopy using canine HAS cells, Abstract;
Genista sessilifolia DC. extracts induce apoptosis across a range of cancer cell lines: P. Bontempo, et al.; Cell Prolif. 46, 183 (2013), Application(s): Detection Kit using live cells, Abstract;
High mobility group box 1 released from necrotic cells enhances regrowth and metastasis of cancer cells that have survived chemotherapy: Y. Luo, et al.; Eur. J. Cancer. 49, 741 (2013), Application(s): Fluorescence microscopy using CT26 cells, Abstract;
Influence of gefitinib and erlotinib on apoptosis and c-MYC expression in H23 lung cancer cells: M. Suenaga, et al.; Anticancer Res. 33, 1547 (2013), Abstract;
Comparison of different suicide-gene strategies for the safety improvement of genetically manipulated T cells: V. Marin, et al.; Hum. Gene Ther. Methods 23, 376 (2012), Application(s): Flow cytometry using EBV-CTL and 293 T cells, Abstract; Full Text
Maintenance of higher H2O2 levels, and its mechanism of action to induce growth in breast cancer cells: Important roles of bioactive catalase and PP2A: S. Sen, et al.; Free Radic. Biol. Med. 53, 1541 (2012), Application(s): Detection Kit using live cells, Abstract;
Mesenchymal stem cells from Shwachman–Diamond syndrome patients display normal functions and do not contribute to hematological defects: V. André, et al.; Blood Cancer J. 2, e94 (2012), Application(s): Flow cytometry using human neutrophils, Abstract; Full Text
Methionine excess in diet induces acute lethal hepatitis in mice lacking cystathionine γ-lyase, an animal model of cystathioninuria: H. Yamada, et al.; Free Radic. Biol. Med. 52, 1716 (2012), Application(s): Fluorescence microscopy using mouse hepatocytes, Abstract;
Molecular mechanism of interleukin-2-induced mucosal homeostasis : J. Mishra, et al.; Am. J. Physiol. Cell Physiol. 302, C735 (2012), Application(s): Detection Kit using live cells, Abstract; Full Text
Serratia marcescens induces apoptotic cell death in host immune cells via a lipopolysaccharide- and flagella-dependent mechanism: K. Ishii, et al.; J. Biol. Chem. 287, 36582 (2012), Application(s): Apoptosis detected in Silkworm larvae, Abstract;
Serratia marcescens Induces Apoptotic Cell Death in Host Immune Cells via a Lipopolysaccharide- and Flagella-dependent Mechanism: K. Ishii, et al.; J. Biol. Chem. 287, 36582 (2012), Application(s): Detection Kit using live cells, Abstract; Full Text
Two mutations impair the stability and function of ubiquitin-activating enzyme (E1): T. Lao, et al.; J. Cell. Physiol. 227, 1561 (2012), Application(s): Viability and induction of cell death observed by confocal microscopy, Abstract; Full Text
Critical roles of Cold Shock Domain Protein A as an endogenous angiogenesis inhibitor in skeletal muscle: Y. Saito, et al.; Antioxid. Redox. Signal. 15, 2109 (2011), Abstract;
Cell Surface Externalization of Annexin A1 as a Failsafe Mechanism Preventing Inflammatory Responses during Secondary Necrosis: K.E. Blume, et al.; J. Immunol. 183, 8138 (2009), Abstract; Full Text
Supplemental Information for: Cell Surface Externalization of Annexin A1 as a Failsafe Mechanism Preventing Inflammatory Responses during Secondary Necrosis: K.E. Blume, et al.; J. Immunol. 183, (2009), (Supplemental Information), Full Text

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