Cell Meter™ PE-Annexin V Binding Apoptosis Assay Kit *Optimized for Flow Cytometry*

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The detection of binding activity of RPE-Annexin V to phosphatidylserine in Jurkat cells with Cell Meter™ RPE-Annexin V Binding Apoptosis Assay Kit. Jurkat cells were treated without (Blue) or with 1 µM staurosporine (Red) in a 37 °C, 5% CO2 incubator for 4-5 hours, and then dye loaded with RPE-Annexin V for 30 minutes. The fluorescence intensity of RPE-Annexin V was measured with a FACSCalibur (Becton Dickinson) flow cytometer using the FL2 channel.
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Unit Size: Cat No: Price (USD): Qty:
100 Tests 22838 $195


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Overview

Ex/Em (nm)565/575
Storage Refrigerated (2-4 °C)
Minimize light exposure
InstrumentsFlow cytometer
Category Cell Analysis
Cell Cytotoxicity
Related Apoptosis and Cytotoxicity
Cell Apoptosis
Annexin V may be conjugated to fluorochromes including PE. This format retains its high affinity for phosphatidylserine (PS) and thus serves as a sensitive probe for flow cytometric analysis of cells that are undergoing apoptosis. Since externalization of PS occurs in the earlier stages of apoptosis, PE Annexin V staining can identify apoptosis at an earlier stage than assays based on nuclear changes such as DNA fragmentation. PE Annexin V staining precedes the loss of membrane integrity which accompanies the latest stages of cell death resulting from either apoptotic or necrotic processes. Therefore, staining with PE Annexin V is typically used in conjunction with a vital dye such as propidium iodide (PI) or 7-Amino-Actinomycin (7-AAD) to allow the investigator to identify early apoptotic cells (7-AAD negative, PE Annexin V positive). Viable cells with intact membranes exclude 7-AAD, whereas the membranes of dead and damaged cells are permeable to 7-AAD. For example, cells that are considered viable are both PE Annexin V and 7-AAD negative while cells that are in early apoptosis are PE Annexin V positive and 7-AAD negative, while cells that are in late apoptosis or already dead are both PE Annexin V and 7-AAD positive. This assay does not distinguish between cells that have undergone apoptotic death versus those that have died as a result of a necrotic pathway because in either case, the dead cells will stain with both PE Annexin V and 7-AAD. However, when apoptosis is measured over time, cells can be often tracked from PE Annexin V and 7-AAD negative (viable, or no measurable apoptosis), to PE Annexin V positive and 7-AAD negative (early apoptosis, membrane integrity is present) and finally to PE Annexin V and 7-AAD positive (end stage apoptosis and death). The movement of cells through these three stages suggests apoptosis. In contrast, a single observation indicating that cells are both PE Annexin V and 7-AAD positive, in of itself, reveals less information about the process by which the cells underwent their demise.




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Protocol


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This protocol only provides a guideline, and should be modified according to your specific needs.
At a glance

Protocol summary

  1. Prepare cells with test compounds (200 µL/sample)
  2. Add RPE-Annexin V assay solution
  3. Incubate at room temperature for 20 - 60 minutes
  4. Analyze cells with a flow cytometer using FL2 channel (Ex/Em = 488/585 nm)
Key parameters
Instrument:Flow cytometer
Instrument specification(s):FL2 channel, FL4 channel
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. RPE-Annexin V stock solution (100X):
Add 200 µL PBS with 0.2% BSA into the vial of RPE-Annexin V (Component A) to make 100X RPE-Annexin V stock solution. Note: Store the reconstituted 100X RPE-Annexin V stock solution at 4°C. Do Not Freeze.

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

Sample experimental protocol
  1. Treat cells with test compounds for a desired period of time (4 - 6 hours for Jurkat cells treated with staurosporine) to induce apoptosis. Note: Annexin V 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 100X RPE-Annexin V stock solution into the cells.

  5. Optional: Add 2 µL of 100X Nuclear Red™ DCS (Component C) for necrosis cells.

  6. Incubate at room temperature for 20 to 60 minutes, protected from light.

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

  8. Monitor the fluorescence intensity of RPE-Annexin V using a flow cytometer with FL2 channel (Ex/Em = 488/585 nm). Measure the cell viability using FL4 channel when Nuclear Red™ DCS  is added into the cells.
Example data analysis and figures

In live non-apoptotic cells, RPE-Annexin V detects innate apoptosis in non-induced cells, which is typically 2- 6% of all cells. In apoptotic cells, RPE-Annexin V binds to phosphatidylserine, which is located on the outer leaflet of the cell membrane, therefore resulted in increased staining intensity.

Figure 1. The detection of binding activity of RPE-Annexin V to phosphatidylserine in Jurkat cells with Cell Meter™ RPE-Annexin V Binding Apoptosis Assay Kit. Jurkat cells were treated without (Blue) or with 1 µM staurosporine (Red) in a 37 °C, 5% CO2 incubator for 4-5 hours, and then dye loaded with RPE-Annexin V for 30 minutes. The fluorescence intensity of RPE-Annexin V was measured with a FACSCalibur (Becton Dickinson) flow cytometer using the FL2 channel.
Disclaimer
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.





References & Citations

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

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

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

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

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, Elstrand MB, Trope CG, Davidson B, Risberg B.
Journal: Am J Clin Pathol (2009): 756

Glycogen synthase kinase-3 and Omi/HtrA2 induce annexin A2 cleavage followed by cell cycle inhibition and apoptosis
Authors: Wang CY, Lin YS, Su WC, Chen CL, Lin CF.
Journal: Mol Biol Cell (2009): 4153

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

Induction of apoptosis in sonoporation and ultrasonic gene transfer
Authors: Miller DL, Dou C.
Journal: Ultrasound Med Biol (2009): 144

Mobilization of lysosomal calcium regulates the externalization of phosphatidylserine during apoptosis
Authors: Mirnikjoo B, Balasubramanian K, Schroit AJ.
Journal: J Biol Chem (2009): 6918

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, Vander Elst L, Corot C, Muller RN.
Journal: Mol Pharm (2009): 1903


View More Citations




Additional Documents

 
Safety Data Sheet (SDS)


Catalogs
1. Cell Apoptosis & Proliferation

Certificate of Analysis