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Cell Meter™ Phosphatidylserine Apoptosis Assay Kit *Blue Fluorescence Excited at 405 nm*

The detection of phosphatidylserine binding activity in Jurkat cells with Cell Meter™ Phosphatidylserine Apoptosis Assay Kit. Jurkat cells were treated without (Blue) or with 1 µM staurosporine (Red) in a 37 °C, 5% CO2 incubator for 5 hours, and then dye loaded with Apopxin™ Violet 450 for 30 minutes. The fluorescence intensity of Apopxin™ Violet 450 was measured with a FACSCalibur (Becton Dickinson) flow cytometer using violet laser.
The detection of phosphatidylserine binding activity in Jurkat cells with Cell Meter™ Phosphatidylserine Apoptosis Assay Kit. Jurkat cells were treated without (Blue) or with 1 µM staurosporine (Red) in a 37 °C, 5% CO2 incubator for 5 hours, and then dye loaded with Apopxin™ Violet 450 for 30 minutes. The fluorescence intensity of Apopxin™ Violet 450 was measured with a FACSCalibur (Becton Dickinson) flow cytometer using violet laser.
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Catalog Number22835
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Telephone1-408-733-1055
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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 for monitoring cell viability. This particular kit is designed to monitor cell apoptosis through measuring the translocation of phosphatidylserine (PS). In apoptosis, PS is transferred to the outer leaflet of the plasma membrane. The appearance of phosphatidylserine on the cell surface is a universal indicator of the initial/intermediate stages of cell apoptosis and can be detected before morphological changes can be observed. Our proprietary Apopxin™ PS sensor used in this kit is small molecule-based PS sensor. The PS sensor has blue fluorescence upon binding to membrane PS. The PS stain used in the kit has the spectral properties similar to those of Pacific Blue® (Pacific Blue® is the trademark of Invitrogen). The blue fluorescent stain is well excited with the Violet Laser at 405 nm, and emits intense blue fluorescence at ~450 nm. The kit is optimized to be used with a flow cytometer equipped with a Violet Laser. It is particularly suitable for multicolor flow cytometric analysis of cells. This kit has been used for flow cytometric analysis of cells in multicolor applications in combination with fluorescent antibodies.

Platform


Flow cytometer

Excitation405 nm laser
Emission450/40 nm filter
Instrument specification(s)Pacific Blue channel

Fluorescence microscope

ExcitationViolet filter
EmissionViolet filter
Recommended plateBlack wall/clear bottom

Components


Component A: Apopxin™ Violet 4501 vial (200 µL)
Component B: Assay Buffer 1 bottle (50 mL)
Component C: 100X Propidium Iodide1 vial (100 µL)

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells with test compounds (200 µL/sample)
  2. Add Apopxin™ Violet 450 assay solution
  3. Incubate at room temperature for 30 - 60 mintues
  4. Analyze cells using flow cytometer with 450/40 nm filter (Pacific Blue channel) or fluorescence microscope with Violet filter

Important notes
Thaw 100X Propidium Iodide (Component C) at room temperature before starting the experiment.

SAMPLE EXPERIMENTAL PROTOCOL

Prepare and incubate cells with Apopxin™ Violet 450:

  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 Apopxin™ Violet 450 (Component A) into the cells.

  5. Optional: Add 2 µL of 100X Propidium Iodide (Component C) into the cells for necrosis cells.

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

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

  8. Monitor the fluorescence intensity using a flow cytometer with 450/40 nm filter (Pacific Blue channel) or a fluorescence microscope with Violet filter.

Analyze by using a flow cytometer:

  1. Quantify Apopxin™ Violet 450 binding using a flow cytometer with 450/40 nm filter (Pacific Blue channel). Measure the cell viability using 610/20 nm filter (PE-Texas Red channel)when propidium iodide is added into the cells. Note: Apopxin™ binding flow cytometric analysis on 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 by 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. Note: For adherent cells, it is recommended to grow the cells directly on a cover-slip. After incubation with Apopxin™ Violet 450, rinse 1 - 2 times with Assay Buffer, and add Assay Buffer back to the cover-slip. 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™ Violet 450 and visualized under a microscope.

  3. Analyze the apoptotic cells with Apopxin™ Violet 450 under a fluorescence microscope with Violet filter. Measure the cell viability using TRITC filter when propidium iodide is added into the cells. The blue staining on the plasma membrane indicates the Apopxin™ Violet 450 binding to PS on cell surface.

Citations


View all 3 citations: Citation Explorer
The Growth, Apoptosis and Oxidative Stress in Microcystis viridis Exposed to Glyphosate
Authors: Ye, Jing and Huang, Chen and Qiu, Zhihao and Wu, Liang and Xu, Chao
Journal: Bulletin of Environmental Contamination and Toxicology (2019): 1--5
Integrin $\beta$3 inhibits hypoxia-induced apoptosis in cardiomyocytes
Authors: Su, Yifan and Tian, Hua and Wei, Lijiang and Fu, Guohui and Sun, Ting
Journal: Acta Biochimica et Biophysica Sinica (2018): 658--665
Integrin β3 inhibits hypoxia-induced apoptosis in cardiomyocytes
Authors: Su, Yifan and Tian, Hua and Wei, Lijiang and Fu, Guohui and Sun, Ting
Journal: Acta Biochimica et Biophysica Sinica (2018)

References


View all 135 references: Citation Explorer
Suicidal membrane repair regulates phosphatidylserine externalization during apoptosis
Authors: Mirnikjoo B, Balasubramanian K, Schroit AJ.
Journal: J Biol Chem (2009): 22512
Peptidic targeting of phosphatidylserine for the MRI detection of apoptosis in atherosclerotic plaques
Authors: Burtea C, Laurent S, Lancelot E, Ballet S, Murariu O, Rousseaux O, Port M, V and er Elst L, Corot C, Muller RN.
Journal: Mol Pharm (2009): 1903
Apoptosis of human Burkitt's lymphoma cells induced by 2-N,N-diethylaminocarbonyloxymethyl-1-diphenylmethyl-4-(3,4,5-trimethoxybe nzoyl) piperazine hydrochloride (PMS-1077)
Authors: Wang WD, Xu XM, Chen Y, Jiang P, Dong CZ, Wang Q.
Journal: Arch Pharm Res (2009): 1727
Induction of apoptosis in sonoporation and ultrasonic gene transfer
Authors: Miller DL, Dou C.
Journal: Ultrasound Med Biol (2009): 144
Detection of apoptosis based on the interaction between annexin V and phosphatidylserine
Authors: Liu T, Zhu W, Yang X, Chen L, Yang R, Hua Z, Li G.
Journal: Anal Chem (2009): 2410
Evaluation of cell surface expression of phosphatidylserine in ovarian carcinoma effusions using the annexin-V/7-AAD assay: clinical relevance and comparison with other apoptosis parameters
Authors: Dong HP, Holth A, Kleinberg L, Ruud MG, Elstr and MB, Trope CG, Davidson B, Risberg B.
Journal: Am J Clin Pathol (2009): 756
Gold fluorescent annexin A5 as a novel apoptosis detection tool
Authors: Kurschus FC, Pal PP, Baumler P, Jenne DE, Wiltschi B, Budisa N.
Journal: Cytometry A (2009): 626
Mobilization of lysosomal calcium regulates the externalization of phosphatidylserine during apoptosis
Authors: Mirnikjoo B, Balasubramanian K, Schroit AJ.
Journal: J Biol Chem (2009): 6918
Eurycomanone induce apoptosis in HepG2 cells via up-regulation of p53
Authors: Zakaria Y, Rahmat A, Pihie AH, Abdullah NR, Houghton PJ.
Journal: Cancer Cell Int (2009): 16
Dynamic analysis of apoptosis using cyanine SYTO probes: from classical to microfluidic cytometry
Authors: Wlodkowic D, Skommer J, Faley S, Darzynkiewicz Z, Cooper JM.
Journal: Exp Cell Res (2009): 1706