Annexin V Assay
Detecting Apoptosis Using Annexin V and NucleoCounter® NC-3000™
- Ascertain if cells are in early or late-stage apoptosis/necrosis
- Fast, automated single cell analysis
- Standardized results – even between different users
- User-friendly protocol with predefined settings
- No calibration required
- Clear data presentation with Plot Manager feature
- Automated PDF reports
- Export data in FCS/ACS formats
Overview of the Annexin V Assay
Externalization, or translocation, of phosphatidylserine (PS) from the inner to the outer membrane leaflet happens in early apoptosis and precedes other apoptotic events such as loss of membrane integrity and DNA fragmentation.
Annexins are a group of cellular proteins that bind to phospholipids in a calcium-dependent manner. A member of this group, Annexin V, has proven to be a useful tool in detecting apoptotic cells since it has a high affinity for PS, and thus translocates with PS to the outer cell membrane.
Using fluorescently labeled Annexin V, the externalization of PS can easily be detected. Annexin V may also bind to PS on late apoptotic and necrotic cells but as the membrane integrity on these cells has been lost, they can be distinguished from early apoptotic cells using propidium iodide (PI). The PI dye is not membrane-permeable and is used to stain cells and DNA.
Assay Principle
Using fluorescence microscopy and image analysis, the NucleoCounter® NC-3000™ advanced image cytometer automatically detects apoptotic cells based on PS externalization. Cells are stained with Hoechst 33342, PI and FITC-labeled Annexin V.
Hoechst 33342 stains the total cell population, while Annexin V stains apoptotic and necrotic cells. Early apoptotic cells exclude PI, while late-stage apoptotic and necrotic cells stain positive for both Annexin V and PI.
Results Presented in Plot Manager
The diagram demonstrates the effect of adding camptothecin (CPT) on Jurkat cells. CPT is a commonly used topoisomerase poison, which induces apoptosis, leading to the externalization of PS and enabling the binding of Annexin V. Jurkat cells are an immortalized line of human T lymphocyte cells, commonly used for studying the effectiveness of potential anti-cancer compounds.
Jurkat cells were grown in the absence (upper row) or presence (lower row) of camptothecin (CPT). Cells were stained with Hoechst 33342, Annexin V-Fluorescein Isothiocyanate (FITC) conjugated antibody and propidium iodide (PI) and analyzed using the Annexin V Assay with the NucleoCounter® NC-3000™.
Scatter plots and histograms were obtained in the NucleoView™ NC-3000™ software. Quadrants (B, F) and markers (C, D, G, H) in the displayed plots were used to demarcate the various cell populations. In this example, CPT causes a significant increase of early apoptotic cells (marked as Annexin V positive and PI negative cells in the lower right quadrant, F).
Tech notes
Publications
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- Instrument: NucleoCounter® NC-3000™
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- Instrument: NucleoCounter® NC-3000™
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- Assay: Annexin V Assay
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- Instrument: NucleoCounter® NC-3000™
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- Instrument: NucleoCounter® NC-3000™
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- Assay: Annexin V Assay
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- Instrument: NucleoCounter® NC-3000™
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- Assay: Annexin V Assay, Cell Count & Viability Assay, Cell Cycle Assay, Cell Vitality Assay, Mitochondrial Potential Assay
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- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: Annexin V Assay
- Research area: Oncology
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- Instrument: NucleoCounter® NC-3000™
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- Assay: Annexin V Assay, Cell Cycle Assay
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103(11):1862-1872
- Instrument: NucleoCounter® NC-3000™
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- Assay: Annexin V Assay, Cell Cycle Assay
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- Instrument: NucleoCounter® NC-3000™
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- Assay: Annexin V Assay
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- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: Annexin V Assay
- Research area: Cell Biology
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2018,
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10(10):e8698.
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Mammalian Cells
- Assay: Annexin V Assay
- Research area: Cell Biology
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Clin Proteomics ,
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2018,
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15.28
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Mammalian Cells
- Assay: Annexin V Assay
- Research area: Cell Biology
Background: Outer membrane vesicles (OMVs) of Acinetobacter baumannii are cytotoxic and elicit a potent innate immune response. OMVs were first identified in A. baumannii DU202, an extensively drug-resistant clinical strain. Herein, we investigated protein components of A. baumannii DU202 OMVs...
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J of Cancer ,
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2017,
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8(8): 1378-1394
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Hepatocytes
- Assay: Annexin V Assay, Cell Cycle Assay
- Research area: Oncology
Hepatocellular carcinoma (HCC) remains one of the most difficult cancers to treat, with chemotherapies being relatively ineffective. Therefore, a better knowledge of molecular hepatocarcinogenesis will provide opportunities for designing targeted therapies. TPX2 (targeting protein for Xklp2) is overexpressed as a...
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Hwang IH, Oh SY, Jang HJ, et al. ,
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2017,
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12(10):e0186489
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: Annexin V Assay
- Research area: Oncology
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Pan HW,, Su HH, et al. ,
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Onco Targets Ther ,
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10:3531-3543
- Instrument: NucleoCounter® NC-3000™
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- Assay: Annexin V Assay, Cell Cycle Assay
- Research area: Oncology
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Huun J, Gansmo LB, Mannsåker B, et al. ,
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Transl Oncol ,
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2017,
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10(5):806-817
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: Annexin V Assay, Cell Cycle Assay, GFP Transfection Efficiency Assay
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Huun J, Gansmo LB, Mannsåker B, et al. ,
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BMC Cell Biol ,
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2017,
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18(1):17
- Instrument: NucleoCounter® NC-3000™
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- Assay: Annexin V Assay, Cell Cycle Assay
- Research area: Oncology
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Kim JM, Hwang IH, Jang IS, et al. ,
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Integr Cancer Ther ,
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2017,
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16(3):360-372
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Hepatocytes
- Assay: Annexin V Assay
- Research area: Oncology
Houttuynia cordata Thunb ( H cordata), a medicinal plant, has anticancer activity, as it inhibits cell growth and induces cell apoptosis in cancer. However, the potential anti-cancer activity and mechanism of H cordata for human liver cancer cells is not...
View PublicationAurora kinase A inhibitor TCS7010 demonstrates pro‑apoptotic effect through the unfolded protein response pathway in HCT116 colon cancer cells
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Lee DH, Chang Gun Kim, Yoongho Lim, Soon Young Shin ,
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Oncology Letters ,
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2017,
-
14(6):6571-6577
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: Annexin V Assay, Cell Cycle Assay
- Research area: Oncology
Aurora kinase A (AURKA) is essential for regulating mitosis and is frequently amplified in various cancer cell types. However, the effect of AURKA inhibition on the induction of apoptosis remains unclear. In the present study, it was reported that treatment...
View PublicationBiological evaluation of 2-pyrazolinyl-1-carbothioamide derivatives against HCT116 humancolorectal cancer cell lines and elucidation on QSAR and molecular binding modes
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Kim BS, Shin SY, Ahn S, et al. ,
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Bioorg Med Chem ,
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2017,
-
25(20):5423-5432
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: Annexin V Assay
- Research area: Oncology
In the search of compounds exhibiting anticancer activity, 37 derivatives of 2-pyrazolinyl-1-carbothioamide were designed and synthesized. Clonogenic cell survival assays were adapted to measure the cytotoxicities of the synthetic derivatives against HCT116 human colon cancer cell lines. Half-maximal cell growth...
View PublicationCordycepin induces human lung cancer cell apoptosis by inhibiting nitric oxide mediatedERK/Slug signaling pathway
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Hwang JH, Park SJ, Ko WG, et al. ,
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Am J Cancer Res ,
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2017,
-
7(3):417-432
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: Annexin V Assay
- Research area: Oncology
Nitric oxide (NO) is an important signaling molecule and a component of the inflammatory cascade. Besides, it is also involved in tumorigenesis. Aberrant upregulation and activation of the ERK cascade by NO often leads to tumor cell development. However, the...
View PublicationSynthesis and biological evaluation of hesperetin derivatives as agents inducing apoptosis
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Jung KY, Park J, Han YS, et al. ,
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Bioorg Med Chem ,
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2017,
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25(1):397-407
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: Annexin V Assay
- Research area: Oncology
A flavanone, hesperetin, has been known to exert antitumor activity by inducing apoptosis. To find hesperetin derivatives showing better antitumor activity, 12 derivatives were designed and synthesized. Their antitumor activities were measured using a long-term survival clonogenic assay. Among the...
View PublicationSenescent cells expose and secrete an oxidized form of membrane-bound vimentin as revealed by a natural polyreactive antibody.
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Frescas D, Roux CM, Aygun-Sunar S, et al. ,
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PNAS ,
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2017,
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114(9):E1668-E1677
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Mammalian Cells
- Assay: Annexin V Assay, Cell Count & Viability Assay
- Research area: Immunology
Studying the phenomenon of cellular senescence has been hindered by the lack of senescence-specific markers. As such, detection of proteins informally associated with senescence accompanies the use of senescence-associated β-galactosidase as a collection of semiselective markers to monitor the presence...
View PublicationCordycepin promotes apoptosis by modulating the ERK-JNK signaling pathway via DUSP5 in renal cancercells.
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Hwang JH, Joo JC, Kim DJ, et al. ,
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Am J Cancer Res ,
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2016,
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6(8):1758-71
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: Annexin V Assay
- Research area: Oncology
The Histone Demethylase PHF8 Is Essential for Endothelial Cell Migration.
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Gu L, Hitzel J,, et al. ,
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PLoS One ,
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2016,
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11(1):e0146645
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Hepatocytes
- Assay: Annexin V Assay
- Research area: Oncology
p53-dependent and -independent mechanisms are involved in (E)-1-(2-hydroxyphenyl)-3-(2-methoxynaphthalen-1-yl)prop-2-en-1-one (HMP)-induced apoptosis in HCT116 colon cancer cells.
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Shin SY, Ahn S, Koh D, Lim Y ,
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Biochem Biophys Res Commun ,
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2016,
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479(4):913-919
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: Annexin V Assay, Cell Cycle Assay
- Research area: Oncology
Small molecule inhibition of Axl receptor tyrosine kinase potently suppresses multiple malignant properties of glioma cells
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Vouri M, Qian An, Matthew Birt, et al. ,
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Oncotarget ,
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2015,
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6(18): 16183–16197
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: Annexin V Assay
- Research area: Oncology
Identification of flavonoids fromEriodictyon californicum and their cytotoxicity against HCT116 colon cancer cells
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Shin SY , Yeonjoong Yong , Dae Sun Hong, et al. ,
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Journal of the Korean Society for Applied Biological Chemistry ,
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2015,
-
77-81
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: Annexin V Assay
- Research area: Oncology
Synthesis of novel C5-curcuminoid-fatty acid conjugates and mechanistic investigation of their anticancer activity
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Sanabria-Ríos DJ, Rivera-Torres Y, Rosario J, et al. ,
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Bioorg Med Chem Lett. ,
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2015,
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pii: S0960-894X(15)00281-4.
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: Annexin V Assay, Mitochondrial Potential Assay
- Research area: Oncology
Enhanced efficacy of chemotherapy for breast cancer stem cells by simultaneous suppression of multidrugresistance and antiapoptotic cellular defense
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Sun M, Yang C, Zheng J, et al. ,
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Acta Biomater ,
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2015,
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28:171-82
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: Annexin V Assay
- Research area: Oncology
Effects of novel alkyl pyridine platinum complexes on apoptosis in Ishikawa endometrial cancer cells.
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Czarnomysy R, Bielawska A, Muszyńska A, Bielawski K ,
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Med Chem ,
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2015,
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11(6):540-50
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: Annexin V Assay
- Research area: Oncology
C5-curcuminoid-4-aminoquinoline based molecular hybrids: design, synthesis and mechanistic investigation of anticancer activity
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Kandi SK, Sunny Manohar, Christian E. Vélez Gerena, et al. ,
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New J Chem ,
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2015,
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39, 224-234
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: Annexin V Assay, DNA Fragmentation Assay
- Research area: Oncology
Hyperphosphatemia induces protective autophagy in endothelial cells through the inhibition of Akt/mTOR signaling
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Hsu YJ, Hsu SC, Huang SM, et al. ,
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J Vasc Surg ,
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2015,
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62(1):210-221
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Hepatocytes
- Assay: Annexin V Assay, FlexiCyte™ Assay
- Research area: Cell Biology
Plant-Derived Flavones as Inhibitors of Aurora B Kinase and Their Quantitative Structure-Activity Relationships.
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Jung Y, Shin SY, Yong Y, et al. ,
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Chem Biol Drug Des. ,
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2015,
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85: 574–585
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Mammalian Cells
- Assay: Annexin V Assay
- Research area: Cell Biology
Mutations in NDUFB11, encoding a complex I component of the mitochondrial respiratory chain, causemicrophthalmia with linear skin defects syndrome
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van Rahden VA, Fernandez-Vizarra E, Alawi M, et al. ,
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Am J Hum Genet ,
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2015,
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96(4):640-50
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: Annexin V Assay
- Research area: Cell Biology
Toxic effects of xylazine on endothelial cells in combination with cocaine and 6-monoacetylmorphine.
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Silva-Torres LA, Vélez C, Lyvia Alvarez J, et al. ,
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Toxicol In Vitro ,
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2014,
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28(7):1312-9
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Whole Blood
- Assay: Annexin V Assay
- Research area: Small Molecule Drugs
Cytocompatibility of direct water synthesized cadmium selenide quantum dots in colo-205 cells
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Rodriguez-Torres MR, Velez C, Zayas B, et al. ,
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J Nanopart Res ,
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2015,
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17(6). pii: 266
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: Annexin V Assay
- Research area: Cell Biology
Proteomic characterization of the outer membrane vesicle of pseudomonas putida KT2440.
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Choi CW, Park EC, Yun SH, et al. ,
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J Proteome Res ,
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2014,
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13(10):4298-309
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Mammalian Cells
- Assay: Annexin V Assay, Cell Count & Viability Assay
- Research area: Small Molecule Drugs
Targeting cancer cells via the reactive oxygen species-mediated unfolded protein response with a novelsynthetic polyphenol conjugate.
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Shin SY, Lee JM, Lee MS, et al. ,
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Clin Cancer Res ,
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2014,
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20(16):4302-13
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: Annexin V Assay, Cell Cycle Assay
- Research area: Oncology
Apoptosis induced by microwave radiation in pancreatic cancer JF305 cells.
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Zhu W, Zhang W, Wang H, et al. ,
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Can J Physiol Pharmacol ,
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2014,
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92(4):324-9
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Hepatocytes
- Assay: Annexin V Assay
- Research area: Oncology
Biological Activity of N-Hydroxyethyl-4-aza- 2,3-didehydropodophyllotoxin Derivatives upon Colorectal Adenocarcinoma Cells
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Vélez C, Zayas B, Kumar A. ,
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Open J Med Chem. ,
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2014,
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4(1):1-11.
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: Annexin V Assay, Cell Cycle Assay, DNA Fragmentation Assay
- Research area: Oncology
Anticancer activity of 4-aminoquinoline-triazine based molecular hybrids
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Manohar S, Antonella Pepe, Christian E. Vélez Gerena, et al. ,
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RSC Advances ,
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2014,
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4(14):7062-7067
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: Annexin V Assay, DNA Fragmentation Assay
- Research area: Oncology
A novel methoxyflavonol derivative, 2-hydroxy-3-(2-methoxyphenyl)-1H-benzo[f]chromen-1-one (DK98), induces apoptosis in HCT116 human colon cancer cells
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Lee MS, Yeonjoong Yong, Jong Min Lee, et al. ,
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Journal of the Korean Society for Applied Biological Chemistry ,
-
2014,
-
(57) 1, pp 129-132
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: Annexin V Assay
- Research area: Oncology
A synthetic naphthochalcone, 2-hydroxy-2,3,4-trimethoxy-5,6-naphthochalcone, triggers caspase-dependent apoptosis in HCT116 human colon cancer cells
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Lee MS, Seunghyun Ahn, Da Hyun Lee, et al. ,
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Journal of the Korean Society for Applied Biological Chemistry ,
-
2014,
-
769-773
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: Annexin V Assay, Cell Cycle Assay
- Research area: Oncology
Imidazolium-derived ionic salts induce inhibition of cancerous cell growth through apoptosis
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Malhotra SV, Vineet Kumar, Christian Velezb and Beatriz Zayasb ,
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Med Chem Comm ,
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2014,
-
5, 1404-1409
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: Annexin V Assay, Cell Cycle Assay, DNA Fragmentation Assay, Mitochondrial Potential Assay
- Research area: Oncology
Comparison of three thiol probes for determination of apoptosis-related changes in cellular redox status.
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Skindersoe ME, Kjaerulff S. ,
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Cytometry A ,
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2014,
-
85(2):179-87
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: Annexin V Assay, FlexiCyte™ Assay, Mitochondrial Potential Assay
- Research area: Cell Biology
Chromenylchalcones showing cytotoxicity on human colon cancer cell lines and in silico docking with aurora kinases
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Shin SY, Yoon H, Ahn S, et al. ,
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Bioorg Med Chem ,
-
2013,
-
21(14):4250-8
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: Annexin V Assay, Cell Cycle Assay
- Research area: Oncology
Down-Regulation of Survivin by Nemadipine-A Sensitizes Cancer Cells to TRAIL-Induced Apoptosis.
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Park SH, Park SJ, Kim JO, et al. ,
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Biomol Ther (Seoul) ,
-
2013,
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21(1)
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Cancer Cells
- Assay: Annexin V Assay
- Research area: Oncology
RNF8 and RNF168 but not HERC2 are required for DNA damage-induced ubiquitylation in chicken DT40 cells
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Oestergaard VH, Pentzold C, Pedersen RT, et al. ,
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DNA Repair ,
-
2012,
-
11(11):892-905
- Instrument: NucleoCounter® NC-3000™
- Cell Type: Mammalian Cells
- Assay: Annexin V Assay, Cell Cycle Assay, FlexiCyte™ Assay
- Research area: Cell Biology