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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.
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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Catalog Number22837
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Spectral properties
Extinction coefficient (cm -1 M -1)700000
Excitation (nm)651
Emission (nm)660
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


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

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

Components


Component A: APC-Annexin V conjugate1 vial
Component B: Assay Buffer (4 °C)1 bottle (50 mL)
Component C: 100X Propidium Iodide1 vial (100 µL)

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells with test compounds (200 µL/sample) 
  2. Add APC-Annexin V assay solution
  3. Incubate at room temperature for 20 - 60 minutes
  4. 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

  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 APC-Annexin V stock solution into the cells.

  5. Optional: Add 2 µL of 100X Propidium Iodide (Component C) into the cells 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 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


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spectrum

Spectral properties

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

Citations


View all 6 citations: Citation Explorer
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
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