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

The detection of binding activity of APC-Annexin V to phosphatidylserine in Jurkat cells with Cell Meter™ APC-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 hours, and then dye loaded with APC-Annexin V for 30 minutes. The fluorescence intensity of APC-Annexin V was measured with a FACSCalibur (Becton Dickinson) flow cytometer using the FL4 channel.

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Spectral properties

Extinction coefficient (cm ^-1 M ^-1)	700000
Excitation (nm)	651
Emission (nm)	660

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

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

700000

Excitation (nm)

651

Emission (nm)

660

Annexin V may be conjugated to fluorochromes including APC. 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, APC Annexin V staining can identify apoptosis at an earlier stage than assays based on nuclear changes such as DNA fragmentation. APC 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 APC 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.

Platform

Flow cytometer

Excitation	640 nm laser
Emission	660/20 nm filter
Instrument specification(s)	APC channel

Components

Example protocol

AT A GLANCE

Protocol summary

Prepare cells with test compounds (200 µL/sample)
Add APC-Annexin V assay solution
Incubate at room temperature for 20 - 60 minutes
Analyze cells using flow cytometer with 660/20 nm filter (APC channel)

Important notes
Thaw 100X Propidium Iodide (Component C) 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. APC-Annexin V stock solution (100X):
Add 200 µL PBS with 0.2% BSA into the vial of APC-Annexin V conjugate (Component A) and mix well to make 100X APC-Annexin V stock solution. Note: Store the reconstituted 100X APC-Annexin V stock solution at 4 ^oC. 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 APC-Annexin V stock solution into the cells.
Optional: Add 2 µL of 100X Propidium Iodide (Component C) into the cells 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 APC-Annexin V using a flow cytometer with 660/20 nm filter (APC channel). Measure the cell viability using 610/20 nm filter (PE-Texas Red channel) when propidium iodide is added into the cells.

Spectrum

Open in Advanced Spectrum Viewer

Spectral properties

Extinction coefficient (cm ^-1 M ^-1)	700000
Excitation (nm)	651
Emission (nm)	660

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Quantum yield	Correction Factor (280 nm)
Cell Meter™ PE-Annexin V Binding Apoptosis Assay Kit Optimized for Flow Cytometry	565	574	1960000	0.82	-
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 APC-Annexin V to phosphatidylserine in Jurkat cells with Cell Meter™ APC-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 hours, and then dye loaded with APC-Annexin V for 30 minutes. The fluorescence intensity of APC-Annexin V was measured with a FACSCalibur (Becton Dickinson) flow cytometer using the FL4 channel.

Citations

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Ubiquitin ligase CHIP functions as an oncogene and activates the AKT signaling pathway in prostate cancer
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Journal: Cell Chemical Biology (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