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

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Spectral properties

Correction Factor (280 nm)	0.175
Extinction coefficient (cm ^-1 M ^-1)	1960000
Excitation (nm)	565
Emission (nm)	574
Quantum yield	0.82

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
UNSPSC	12352200

Overview SDS Protocol

Correction Factor (280 nm)

0.175

Extinction coefficient (cm ^-1 M ^-1)

1960000

Excitation (nm)

565

Emission (nm)

574

Quantum yield

0.82

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.

Platform

Flow cytometer

Excitation	488 nm or 561 nm laser
Emission	575/26 nm filter
Instrument specification(s)	PE channel

Components

Example protocol

AT A GLANCE

Protocol summary

Prepare cells with test compounds (200 µL/sample)
Add RPE-Annexin V assay solution
Incubate at room temperature for 20 - 60 minutes
Analyze cells with a flow cytometer using 575/26 nm filter (PE 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

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.
Centrifuge the cells to get 1 - 5 × 10⁵ cells/tube.
Resuspend cells in 200 µL of Assay Buffer (Component B).
Add 2 µL of 100X RPE-Annexin V stock solution into the cells.
Optional: Add 2 µL of 100X Nuclear Red™ DCS (Component C) for necrosis cells.
Incubate at room temperature for 20 to 60 minutes, protected from light.
Optional: add 200 to 300 µL of Assay Buffer (Component B) to increase volume before analyzing the cells with a flow cytometer.
Monitor the fluorescence intensity of RPE-Annexin V using a flow cytometer with 575/26 nm filter (PE channel). Measure the cell viability using 660/20 nm filter (APC channel) when Nuclear Red™ DCS is added into the cells.

Spectrum

Open in Advanced Spectrum Viewer

Spectral properties

Correction Factor (280 nm)	0.175
Extinction coefficient (cm ^-1 M ^-1)	1960000
Excitation (nm)	565
Emission (nm)	574
Quantum yield	0.82

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Quantum yield	Correction Factor (280 nm)
Cell Meter™ APC-Annexin V Binding Apoptosis Assay Kit Optimized for Flow Cytometry	651	660	730000	-	0.195
Cell Meter™ FITC-Annexin V Binding Apoptosis Assay Kit Optimized for Flow Cytometry	491	516	73000	0.92	0.35

Images

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.

Citations

View all 1 citations: Citation Explorer

Heparanase induces necroptosis of microvascular endothelial cells to promote the metastasis of hepatocellular carcinoma
Authors: Chen, Xiaopeng and Cheng, Bin and Dai, Dafei and Wu, Yuhai and Feng, Zhiwen and Tong, Chaogang and Wang, Xiangming and Zhao, Jun
Journal: Cell death discovery (2021): 1--13

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