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Cell Meter™ Apoptotic and Necrotic Multiplexing Detection Kit II *Triple Fluorescence Colors*

The detection of binding activity of Apopxin™ Deep Red to phosphatidylserine in Jurkat cells using Cell Meter™ Apoptotic and Necrotic Mulptiplexing Detection Kit II. The fluorescence images showing cells that are live (blue, stained by CytoCalcein™ Violet 450), apoptotic (red, stained by Apopxin™ Deep Red), and necrotic (green, indicated by Nuclear Green™ DCS1 staining) in Jurkat cells induced by 1 μM staurosporine for 3 hours. The fluorescence images of the cells were taken with Olympus fluorescence microscope through the Violet, Cy5 and FITC channel respectively. Individual images taken from each channel from the same cell population were merged as shown above. A: Non-induced control cells; B: Triple staining of staurosporine-induced cells.
The detection of binding activity of Apopxin™ Deep Red to phosphatidylserine in Jurkat cells using Cell Meter™ Apoptotic and Necrotic Mulptiplexing Detection Kit II. The fluorescence images showing cells that are live (blue, stained by CytoCalcein™ Violet 450), apoptotic (red, stained by Apopxin™ Deep Red), and necrotic (green, indicated by Nuclear Green™ DCS1 staining) in Jurkat cells induced by 1 μM staurosporine for 3 hours. The fluorescence images of the cells were taken with Olympus fluorescence microscope through the Violet, Cy5 and FITC channel respectively. Individual images taken from each channel from the same cell population were merged as shown above. A: Non-induced control cells; B: Triple staining of staurosporine-induced cells.
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Catalog Number22843
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Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
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ShippingStandard overnight for United States, inquire for international
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H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200
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OverviewpdfSDSpdfProtocol


Our Cell Meter™ assay kits are a set of tools for monitoring cell viability. There are a variety of parameters that can be used. This particular kit is designed to simultaneously monitor apoptotic, necrotic and healthy cells. Apoptosis is described as an active, programmed process of autonomous cellular dismantling that avoids eliciting inflammation. In apoptosis, phosphatidylserine (PS) is transferred to the outer leaflet of the plasma membrane. As a universal indicator of the initial/intermediate stages of cell apoptosis, the appearance of phosphatidylserine on the cell surface can be detected before morphological changes are observed. The PS sensor used in this kit has red fluorescence upon binding to membrane PS. Necrosis has been characterized as passive, accidental cell death resulting from environmental perturbations with uncontrolled release of inflammatory cellular contents. Loss of plasma membrane integrity, as demonstrated by the ability of a membrane-impermeable DNA Nuclear Green™ DCS1 (Ex/Em = 490/525 nm) to label the nucleus, represents a straightforward approach to demonstrate late stage apoptosis and necrosis. In addition, this kit also provides a live cell cytoplasm labeling dye CytoCalcein™ Violet 450 (Ex/Em = 405/450 nm) for labeling living cell cytoplasm. This kit is optimized to simultaneously detect cell apoptosis (Red), necrosis (green and/or red) and healthy cells (blue) with a flow cytometer or fluorescence microscope.

Platform


Flow cytometer

Excitation405 nm, 488 nm, 633 nm laser
Emission450/40 nm, 530/30 nm, 660/20 nm filter
Instrument specification(s)Pacific Blue, FITC, APC channel

Fluorescence microscope

ExcitationDAPI, FITC, Cy5 filter sets
EmissionDAPI, FITC, Cy5 filter sets
Recommended plateBlack wall/clear bottom

Components


Component A: 100X Apopxin™ Deep Red1 vial (200 µL)
Component B: Assay Buffer1 bottle (50 mL)
Component C: 200X Nuclear Green™ DCS11 vial (100 µL)
Component D: CytoCalcein™ Violet 4501 vial (lyophilized powder)

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells with test compounds (200 µL/sample)
  2. Add Apopxin™ Deep Red assay solution
  3. Incubate at room temperature for 30 - 60 minutes
  4. Analyze cells with a flow cytometer with 660/20 nm (for apotosis-APC channel), 530/30 nm (for necrosis-FITC channel) and 450/40 nm emission filter (for healthy cells-Pacific Blue channel) or fluorescence microscope with DAPI (Healthy cells), FITC (Necrosis cells) and Cy5 filters (Apoptotic cells)

Important notes
Thaw all the kit components at room temperature before starting the experiment.

PREPARATION OF STOCK SOLUTION

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. CytoCalcein™ Violet 450 stock solution (200X):
Add 100 µL of DMSO into the vial of CytoCalcein™ Violet 450 (Component D) to make 200X CytoCalcein™ Violet 450 stock solution. Protect from light.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

SAMPLE EXPERIMENTAL PROTOCOL

Prepare and incubate cells with Apopxin™ Deep Red:

  1. Treat cells with test compounds for a desired period of time (4-6 hours for Jurkat cells treated with staurosporine) to induce apoptosis.

  2. Centrifuge the cells to get 1-5×105 cells/tube.

  3. Resuspend cells in 200 µL of Assay Buffer (Component B).

  4. Add 2 µL of 100X Apopxin™ Deep Red (Component A) into the cells. 

  5. Optional 1: Add 1 µL of 200X Nuclear Green™ DCS1 (Component C) into the cells for necrosis cells.

  6. Optional 2: Then add 1 µL of 200X CytoCalcein™ Violet 450 stock solution into the cells for healthy cells staining.

  7. Incubate at room temperature for 30 to 60 minutes, protected from light.

  8. Add 300 µL of Assay Buffer (Component B) to increase volume before analyzing the cells with a flow cytometer or fluorescence microscope.

  9. Monitor the fluorescence intensity using a flow cytometer with 660/20 nm (for apotosis-APC channel), 530/30 nm (for necrosis-FITC channel) and 450/40 nm emission filter (for healthy cells-Pacific Blue channel) or fluorescence microscope with DAPI (Healthy cells), FITC (Necrosis cells) and Cy5 filters (Apoptotic cells).

Analyze cells using a flow cytometer:

  1. Quantify Apopxin™ Deep Red binding using a flow cytometer with 660/20 nm (for apotosis-APC channel), 530/30 nm (for necrosis-FITC channel) and 450/40 nm emission filter (for healthy cells-Pacific Blue channel). Note: The flow cytometric analysis of Apopxin™ binding to adherent cells is not routinely tested since specific membrane damage may occur during cell detachment or harvesting. However, methods for utilizing Annexin V for flow cytometry on adherent cell types have been previously reported by Casiola-Rosen et al. and van Engelend et al.

 Analyze cells using a fluorescence microscope:

  1. Pipette the cell suspension after incubation, rinse 1-2 times with Assay Buffer, and then resuspend the cells with Assay Buffer.

  2. Add the cells on a glass slide that is covered with a glass cover-slip or a black wall/clear bottom 96-well microplate. Note: For adherent cells, it is recommended to grow the cells directly on a cover-slip (or a black wall/clear bottom 96-well microplate). After incubation with Apopxin™ Deep Red, rinse 1-2 times with assay buffer, and then add assay buffer back to the cover-slip (or a black wall/clear bottom 96-well microplate). Invert cover-slip on a glass slide and visualize the cells. The cells can also be fixed in 2% formaldehyde after the incubation with Apopxin™ Deep Red and visualized under a microscope.

  3. Analyze the apoptotic cells with Apopxin™ Deep Red under a fluorescence microscope using Cy5 filter. Measure the cell viability using FITC filter when Nuclear Green™ DCS1 is added, and/or Violet filter when CytoCalcein™ Violet 450 is added into the cells. The red staining on the plasma membrane indicates the Apopxin™ Deep Red binding to PS on cell surface.

Citations


View all 4 citations: Citation Explorer
Dasatinib can Impair Left Ventricular Mechanical Function But May Lack Proarrhythmic Effect: A Proposal of Non-clinical Guidance for Predicting Clinical Cardiovascular Adverse Events of Tyrosine Kinase Inhibitors
Authors: Izumi-Nakaseko, Hiroko and Fujiyoshi, Masachika and Hagiwara-Nagasawa, Mihoko and Goto, Ai and Chiba, Koki and Kambayashi, Ryuichi and Naito, Atsuhiko T and Ando, Kentaro and K, undefined and a, Yasunari and Ishii, Itsuko and others, undefined
Journal: Cardiovascular Toxicology (2019): 1--13
Anthocyanin-rich blackcurrant extract inhibits proliferation of the MCF10A healthy human breast epithelial cell line through induction of G0/G1 arrest and apoptosis
Authors: Nanashima, Naoki and Horie, Kayo and Chiba, Mitsuru and Nakano, Manabu and Maeda, Hayato and Nakamura, Toshiya
Journal: Molecular Medicine Reports (2017): 6134--6141
Clusterin signals via ApoER2/VLDLR and induces meiosis of male germ cells
Authors: Riaz, Muhammad Assad and Stammler, Angelika and Borgers, Mareike and Konrad, Lutz
Journal: American Journal of Translational Research (2017): 1266
Detecting Apoptosis, Autophagy, and Necrosis
Authors: Coleman, Jack and Liu, Rui and Wang, Kathy and Kumar, Arun
Journal: Apoptosis Methods in Toxicology (2016): 77--92

References


View all 71 references: Citation Explorer
Synergistic effects and mechanisms of combined tumor necrosis factor-related apoptosis-inducing ligand and chemotherapeutic drugs or radiotherapy in killing laryngeal squamous carcinoma cells in vitro
Authors: Zhang M, Zhou L.
Journal: Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi (2009): 565
Down-regulation of myeloid cell leukemia 1 by epigallocatechin-3-gallate sensitizes rheumatoid arthritis synovial fibroblasts to tumor necrosis factor alpha-induced apoptosis
Authors: Ahmed S, Silverman MD, Marotte H, Kwan K, Matuszczak N, Koch AE.
Journal: Arthritis Rheum (2009): 1282
Agmatine protects cultured retinal ganglion cells from tumor necrosis factor-alpha-induced apoptosis
Authors: Hong S, Kim CY, Lee JE, Seong GJ.
Journal: Life Sci (2009): 28
Exaggerated up-regulation of tumor necrosis factor alpha-dependent apoptosis in the older mouse liver following reperfusion injury: targeting liver protective strategies to patient age
Authors: Selzner M, Selzner N, Chen L, Borozan I, Sun J, Xue-Zhong M, Zhang J, McGilvray ID.
Journal: Liver Transpl (2009): 1594
Oxidant stress-induced liver injury in vivo: role of apoptosis, oncotic necrosis, and c-Jun NH2-terminal kinase activation
Authors: Hong JY, Lebofsky M, Farhood A, Jaeschke H.
Journal: Am J Physiol Gastrointest Liver Physiol (2009): G572
A pharmaceutical preparation of Salvia miltiorrhiza protects cardiac myocytes from tumor necrosis factor-induced apoptosis and reduces angiotensin II-stimulated collagen synthesis in fibroblasts
Authors: Ling S, Luo R, Dai A, Guo Z, Guo R, Komesaroff PA.
Journal: Phytomedicine (2009): 56
Increased apoptosis in HepG2.2.15 cells with hepatitis B virus expression by synergistic induction of interferon-gamma and tumour necrosis factor-alpha
Authors: Shi H, Guan SH.
Journal: Liver Int (2009): 349
Outside-to-inside signaling through transmembrane tumor necrosis factor reverses pathologic interleukin-1beta production and deficient apoptosis of rheumatoid arthritis monocytes
Authors: Meusch U, Rossol M, Baerwald C, Hauschildt S, Wagner U.
Journal: Arthritis Rheum (2009): 2612
Tumor necrosis factor inhibitors block apoptosis of human epithelial cells of the salivary glands
Authors: Sisto M, D'Amore M, Caprio S, Mitolo V, Scagliusi P, Lisi S.
Journal: Ann N Y Acad Sci (2009): 407
Susceptibility of the C2 canine mastocytoma cell line to the effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)
Authors: Elders RC, Baines SJ, Catchpole B.
Journal: Vet Immunol Immunopathol (2009): 11