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Cell Meter™ Phosphatidylserine Apoptosis Assay Kit *Deep Red Fluorescence Optimized for Flow Cytometry*

Detection of phosphatidylserine binding activity in Jurkat cells. Jurkat cells were treated without (Blue) or with 20 µM camptothecin (Red) in a 37 °C, 5% CO2 incubator for 4-5 hours, and then loaded with Apopxin™ Deep Red for 30 minutes. The fluorescence intensity of Apopxin™ Deep Red was measured with a FACSCalibur (Becton Dikinson) flow cytometer in FL4 channel.
Detection of phosphatidylserine binding activity in Jurkat cells. Jurkat cells were treated without (Blue) or with 20 µM camptothecin (Red) in a 37 °C, 5% CO2 incubator for 4-5 hours, and then loaded with Apopxin™ Deep Red for 30 minutes. The fluorescence intensity of Apopxin™ Deep Red was measured with a FACSCalibur (Becton Dikinson) flow cytometer in FL4 channel.
Detection of phosphatidylserine binding activity in Jurkat cells. Jurkat cells were treated without (Blue) or with 20 µM camptothecin (Red) in a 37 °C, 5% CO2 incubator for 4-5 hours, and then loaded with Apopxin™ Deep Red for 30 minutes. The fluorescence intensity of Apopxin™ Deep Red was measured with a FACSCalibur (Becton Dikinson) flow cytometer in FL4 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 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. It has red fluorescence upon binding to membrane PS. This particular assay kit is optimized to monitor cell apoptosis using a flow cytometer at Cy5 channel (red fluorescence).

Platform


Flow cytometer

Excitation640 nm laser
Emission660/20 nm filter
Instrument specification(s)APC channel

Components


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 using flow cytometer with FL4 channel (Ex/Em = 647/660 nm

SAMPLE EXPERIMENTAL PROTOCOL

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

  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™ Deep Red (Component A) into the cells.

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

  6. Optional: add 200 to 300 µL of Assay Buffer (Component B) to increase volume before analyzing the cells with a flow cytometer.

  7. Monitor the fluorescence intensity using a flow cytometer with FL4 channel (Ex/Em = 647/660 nm).

Images


Citations


View all 2 citations: Citation Explorer
STE20-Type Kinases MST3 and MST4 Act Non-Redundantly to Promote the Progression of Hepatocellular Carcinoma
Authors: Caputo, Mara and Xia, Ying and Anand, Sumit Kumar and Cansby, Emmelie and Andersson, Emma and Marschall, Hanns-Ulrich and K{\"o}nigsrainer, Alfred and Peter, Andreas and Mahlapuu, Margit
Journal: (2023)
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

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