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

The fluorescence images showing cells that are live (blue, stained by CytoCalcein™ Violet 450), apoptotic (green, stained by Apopxin™ Green), and necrotic (red, indicated by 7-AAD staining) in Jurkat cells induced by 1µM staurosporine for 3 hours. The fluorescence images of the cells were taken with Olympus fluorescence microscope through the Violet, FITC and Texas Red channel respectively. Individual images taken from each channel from the same cell population were merged as shown above. Left: Non-induced control cells; Right: Triple staining of staurosporine-induced cells.
The fluorescence images showing cells that are live (blue, stained by CytoCalcein™ Violet 450), apoptotic (green, stained by Apopxin™ Green), and necrotic (red, indicated by 7-AAD staining) in Jurkat cells induced by 1µM staurosporine for 3 hours. The fluorescence images of the cells were taken with Olympus fluorescence microscope through the Violet, FITC and Texas Red channel respectively. Individual images taken from each channel from the same cell population were merged as shown above. Left: Non-induced control cells; Right: Triple staining of staurosporine-induced cells.
The fluorescence images showing cells that are live (blue, stained by CytoCalcein™ Violet 450), apoptotic (green, stained by Apopxin™ Green), and necrotic (red, indicated by 7-AAD staining) in Jurkat cells induced by 1µM staurosporine for 3 hours. The fluorescence images of the cells were taken with Olympus fluorescence microscope through the Violet, FITC and Texas Red channel respectively. Individual images taken from each channel from the same cell population were merged as shown above. Left: Non-induced control cells; Right: Triple staining of staurosporine-induced cells.
The detection of binding activity of Apopxin&trade; Green to phosphatidylserine in Jurkat cells. Jurkat cells were treated without (A. Blue) or with 1 &mu;M staurosporine (A. Red) in a 37 <sup>o</sup>C, 5% CO<sub>2</sub> incubator for 5 hours, and then loaded with Apopxin&trade; Green for 30 minutes. The fluorescence intensity of Apopxin&trade; Green was measured with a FACSCalibur (Becton Dickinson, San Jose, CA) flow cytometer using FL1 channel.
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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 monitor cell 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 green 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 7-AAD (Ex/Em = 546/647 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 detect cell apoptosis (green), necrosis (green and/or red) and healthy cells (blue) with a flow cytometer and fluorescence microscope.

Platform


Flow cytometer

Excitation405 nm, 488 nm laser
Emission450/40 nm, 530/30 nm, 670/14 nm filter
Instrument specification(s)Pacific Blue, FITC, PE-Cy5 channel

Fluorescence microscope

ExcitationDAPI, FITC, Texas Red filter
EmissionDAPI, FITC, Texas Red filter
Recommended plateBlack wall/clear bottom

Components


Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells with test compounds (200 µL/sample)
  2. Add Apopxin™ Green assay solution
  3. Incubate at room temperature for 30 - 60 minutes
  4. Analyze cells with a flow cytometer with emission filter 530/30 nm (FITC channel- for apoptotic cells), 450/40 nm (Pacific Blue channel- for healthy cells) and 670/14 nm (PE-Cy5 channel- for necrotic 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™ Green:

  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™ Green (Component A) into the cells. 

  5. Optional 1: Add 1 µL of 200X 7-AAD (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 emission filter 530/30 nm (FITC channel- for apoptotic cells), 450/40 nm (Pacific Blue channel- for healthy cells) and 670/14 nm (PE-Cy5 channel- for necrotic cells).

Analyze cells using a flow cytometer:

  1. Quantify Apopxin™ Green binding using a flow cytometer with emission filter 530/30 nm (FITC channel- for apoptotic cells), 450/40 nm (Pacific Blue channel- for healthy cells) and 670/14 nm (PE-Cy5 channel- for necrotic cells). 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™ Green, 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™ Green and visualized under a microscope.

  3. Analyze the apoptotic cells with Apopxin™ Green under a fluorescence microscope using the FITC filter. Measure the cell viability using Texas Red filter when 7-AAD is added, and/or DAPI or Violet filter when CytoCalcein™ Violet 450 is added into the cells. The green staining on the plasma membrane indicates the Apopxin™ Green binding to PS on cell surface.

Images


Citations


View all 7 citations: Citation Explorer
Co-expression of SLC20A1 and ALDH1A3 is associated with poor prognosis, and SLC20A1 is required for the survival of ALDH1-positive pancreatic cancer stem cells
Authors: Matsuoka, Izumi and Kasai, Takahiro and Onaga, Chotaro and Ozaki, Ayaka and Motomura, Hitomi and Maemura, Yuki and Tada, Yuna and Mori, Haruka and Hara, Yasushi and Xiong, Yuyun and others,
Journal: Oncology Letters (2024): 1--16
Mesenchymal Stem Cells Preconditioned with Hypoxia and Dexamethasone Promote Osteoblast Differentiation Under Stress Conditions
Authors: Shimasaki, Miyako and Ichiseki, Toru and Ueda, Shusuke and Hirata, Hiroaki and Kawahara, Norio and Ueda, Yoshimichi
Journal: International Journal of Medical Sciences (2024): 1511--1517
Transcriptional coregulator Ess2 controls survival of post-thymic CD4+ T cells through the Myc and IL-7 signaling pathways
Authors: Takada, Ichiro and Hidano, Shinya and Takahashi, Sayuri and Yanaka, Kaori and Ogawa, Hidesato and Tsuchiya, Megumi and Yokoyama, Atsushi and Sato, Shingo and Ochi, Hiroki and Nakagawa, Tohru and others,
Journal: Journal of Biological Chemistry (2022)
Acrylonitrile Butadiene Styrene (ABS) and Polycarbonate (PC) Filaments Three-Dimensional (3-D) Printer Emissions-Induced Cell Toxicity
Authors: Farcas, Mariana T and Stefaniak, Aleks and r B , undefined and Knepp, Alycia K and Bowers, Lauren and M, undefined and ler, William K and Kashon, Michael and Jackson, Stephen R and Stueckle, Todd A and Sisler, Jenifer D and Friend, Sherri A and others, undefined
Journal: Toxicology Letters (2019)
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
Rapid simultaneous determination of apoptosis, necrosis, and viability in sulfur mustard exposed HaCaT cell cultures
Authors: Heinrich A, Balszuweit F, Thiermann H, Kehe K.
Journal: Toxicol Lett (2009): 260
PVP-coated silver nanoparticles and silver ions induce reactive oxygen species, apoptosis and necrosis in THP-1 monocytes
Authors: Foldbjerg R, Olesen P, Hougaard M, Dang DA, Hoffmann HJ, Autrup H.
Journal: Toxicol Lett (2009): 156
Concurrent induction of necrosis, apoptosis, and autophagy in ischemic preconditioned human livers formerly treated by chemotherapy
Authors: Domart MC, Esposti DD, Sebagh M, Olaya N, Harper F, Pierron G, Franc B, Tanabe KK, Debuire B, Azoulay D, Brenner C, Lemoine A.
Journal: J Hepatol (2009): 881
RAS mutations affect tumor necrosis factor-induced apoptosis in colon carcinoma cells via ERK-modulatory negative and positive feedback circuits along with non-ERK pathway effects
Authors: Kreeger PK, M and hana R, Alford SK, Haigis KM, Lauffenburger DA.
Journal: Cancer Res (2009): 8191
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
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
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
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
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
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