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

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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	0.16
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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Upregulation of LPGAT1 Enhances Lung Adenocarcinoma Proliferation
Authors: Gong, Huiyuan and Ma, Chao and Li, Xiaojun and Zhang, Xueying and Zhang, Linxiang and Chen, Pengfei and Wang, Wei and Hu, Yannan and Huang, Ting and Wu, Nan and others,
Journal: Frontiers in Bioscience-Landmark (2023): 89

BRAF D594A mutation defines a unique biological and immuno-modulatory subgroup associated with functional CD8+ T cell infiltration in colorectal cancer
Authors: Li, Wenjing and Zhao, Chenyi and Li, Wenhui and Gong, Yang and Ma, Kaili and Lu, Yujie and Liu, Xiaowei and Zhang, Lianjun and Guo, Feng
Journal: (2023)

Th22/IL-22 mediates the progression of HBV-related hepatocellular carcinoma via STAT3
Authors: Zhang, Jia and Liu, Zhou and Liu, Lingpeng and Huang, Mingwen and Huang, Yong
Journal: Cytotechnology (2022): 203--216

Effects of Y-27632 on the osteogenic and adipogenic potential of human dental pulp stem cells in vitro
Authors: Xie, Zhiwei and Xu, Qiuping and Sun, Lu and Li, Ruijing and Shi, Jizhou and Yang, Qian and Zong, Min and Qin, Jianyong
Journal: Human \& Experimental Toxicology (2022): 09603271221089003

Knockdown of CENPK inhibits cell growth and facilitates apoptosis via PTEN-PI3K-AKT signalling pathway in gastric cancer
Authors: Wu, Shusheng and Cao, Lulu and Ke, Lihong and Yan, Ying and Luo, Huiqin and Hu, Xiaoxiu and Niu, Jiayu and Li, Huimin and Xu, Huijun and Chen, Wenju and others,
Journal: Journal of Cellular and Molecular Medicine (2021): 8890--8903

FIGNL1 promotes non-small cell lung cancer cell proliferation
Authors: Li, Miao and Rui, Yan and Peng, Wenjia and Hu, Junfeng and Jiang, Anbang and Yang, Zeyu and Huang, Linian
Journal: International journal of oncology (2020): 83--99

Babao Dan induces gastric cancer cell apoptosis via regulating MAPK and NF-$\kappa$B signaling pathways
Authors: Shang, Haixia and Cao, Zhiyun and Zhao, Jinyan and Guan, Jianhua and Liu, Jianxin and Peng, Jun and Chen, Youqin and Joseph Sferra, Thomas and Sankararaman, Senthilkumar and Lin, Jiumao
Journal: Journal of International Medical Research (2019): 5106--5119

CHIP functions as an oncogene by promoting colorectal cancer metastasis via activation of MAPK and AKT signaling and suppression of E-cadherin
Authors: Xu, Jingjing and Zhou, Jun and Dai, Hanjue and Liu, Fei and Li, Wenjing and Wang, Wenjuan and Guo, Feng
Journal: Journal of Translational Medicine (2018): 169

Ubiquitin ligase CHIP functions as an oncogene and activates the AKT signaling pathway in prostate cancer
Authors: Cheng, Li and Zang, Jin and Dai, Han-Jue and Li, Feng and Guo, Feng
Journal: International journal of oncology (2018)

ALDH1 Bio-activates Nifuroxazide to Eradicate ALDHHigh Melanoma-Initiating Cells
Authors: Sarvi, Sana and Crispin, Richard and Lu, Yuting and Zeng, Lifan and Hurley, Thomas D and Houston, Douglas R and von Kriegsheim, Alex and Chen, Che-Hong and Mochly-Rosen, Daria and Ranzani, Marco and others, undefined
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
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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
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Journal: Am J Clin Pathol (2009): 756

Gold fluorescent annexin A5 as a novel apoptosis detection tool
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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
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Journal: Cancer Cell Int (2009): 16

Dynamic analysis of apoptosis using cyanine SYTO probes: from classical to microfluidic cytometry
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