logo
AAT Bioquest

Cell Meter™ Annexin V Binding Apoptosis Assay Kit *Orange Fluorescence Excited at 405 nm*

Our Cell Meter™ assay kits are a set of tools for monitoring cell viability. There are a variety of parameters that can be used for monitoring cell viability. This particular kit is designed to monitor cell apoptosis through measuring the translocation of phosphatidylserine (PS). In apoptosis, PS is transferred to the outer leaflet of the plasma membrane. The appearance of phosphatidylserine on the cell surface is a universal indicator of the initial/intermediate stages of cell apoptosis and can be detected before morphological changes can be observed. This kit uses a fluorescent Annexin V that specifically binds PS. Annexin V conjugates have been demonstrated to selectively bind PS. This particular assay kit is optimized to monitor cell apoptosis using a flow cytometer with the Pacific Orange filter set.

Example protocol

AT A GLANCE

Protocol summary

  1. Prepare cells with test compounds (200 µL/sample)
  2. Add Annexin V-mFluor Violet™ 550 assay solution
  3. Incubate at room temperature for 30 - 60 mintues
  4. Analyze cells using flow cytometer with 525/50 nm filter (Pacific Orange channel) or fluorescence microscope with Violet filter set

Important notes
Thaw 100X Propidium Iodide (Component C) at room temperature before starting the experiment.

SAMPLE EXPERIMENTAL PROTOCOL

Prepare and incubate cells with Annexin V-mFluor Violet™ 550:

  1. Treat cells with test compounds for a desired period of time (4 - 6 hours for Jurkat cells treated with staurosporine) to induce apoptosis.

  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 Annexin V-mFluor Violet™ 550 (Component A) 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 30 to 60 minutes, protected from light.

  7. Add 300 µL of Assay Buffer (Component B) to increase volume before analyzing the cells with a flow cytometer or fluorescence microscope.

  8. Monitor the fluorescence intensity using a flow cytometer with 525/50 nm filter (Pacific Orange channel) or a fluorescence microscope with Violet filter set.

Analyze by using a flow cytometer:

  1. Quantify Annexin V-mFluor Violet™ 550 binding using a flow cytometer with 525/50 nm filter (Pacific Orange channel). Measure the cell viability using 610/20 nm filter (PE-Texas Red channel) when propidium iodide is added into the cells. Note: Annexin V binding 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.

Analyze by using a fluorescence microscope:

  1. Pipette the cell suspension after incubation, rinse 1 - 2 times with assay buffer, and then resuspend the cells with assay buffer.

  2. Add the cells on a glass slide that is covered with a glass cover-slip. Note: For adherent cells, it is recommended to grow the cells directly on a cover-slip. After incubation with Annexin V-mFluor Violet™ 550, rinse 1 - 2 times with assay buffer, and add assay buffer back to the cover-slip. Invert cover-slip on a glass slide and visualize the cells. The cells can also be fixed in 2% formaldehyde after the incubation with Annexin V-mFluor Violet™ 550 and visualized under a microscope.

  3. Analyze the apoptotic cells with Annexin V-mFluor Violet™ 550 under a fluorescence microscope using Violet filter set. Measure the cell viability using TRITC channel when propidium iodide is added into the cells. The orange staining on the plasma membrane indicates the Annexin V-mFluor Violet™ 550 binding to PS on cell surface.

Spectrum

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
Cell Meter™ Annexin V Binding Apoptosis Assay Kit *Blue Fluorescence Excited at 405 nm*4064453500010.8110.3380.078
Cell Meter™ Annexin V Binding Apoptosis Assay Kit *Green Fluorescence Excited at 405 nm*4105012500010.8110.7690.365

Citations

View all 7 citations: Citation Explorer
MiR-934 Exacerbates Malignancy of Gastric Cancer Cells by Targeting ZFP36
Authors: Pan, Zhicheng and Yun, Huazhong and Xiao, Yun and Tong, Fei and Liu, Guodong and Zhang, Ge and Han, Jianbo
Journal: Iranian Journal of Public Health (2023): 1720--1729
CCT6A knockdown suppresses osteosarcoma cell growth and Akt pathway activation in vitro
Authors: Zeng, Weiquan and Wu, Meizhu and Cheng, Ying and Liu, Liya and Han, Yuying and Xie, Qiurong and Li, Jiapeng and Wei, Lihui and Fang, Yi and Chen, Youqin and others,
Journal: Plos one (2022): e0279851
In situ polymerizable hydrogel incorporated with specific pathogen-free porcine platelet-rich plasma for the reconstruction of the corneal endothelium
Authors: Lin, Yung-Kai and Sharma, Ruchi and Ma, Hsu and Chen, Wen-Shyan and Yao, Chao-Ling
Journal: Journal of the Taiwan Institute of Chemical Engineers (2017)
Novel regulations of MEF2-A, MEF2-D, and CACNA1S in the functional incompetence of adipose-derived mesenchymal stem cells by induced indoxyl sulfate in chronic kidney disease
Authors: Do, Duyen Thi and Phan, Nam Nhut and Wang, Chih-Yang and Sun, Zhengda and Lin, Yen-Chang
Journal: Cytotechnology (2016): 2589--2604
Shear stress-induced alteration of epithelial organization in human renal tubular cells
Authors: Maggiorani, Damien and Dissard, Romain and Belloy, Marcy and Saulnier-Blache, Jean-S{\'e}bastien and Casemayou, Audrey and Ducasse, Laure and Gr{\`e}s, Sandra and Belli{\`e}re, Julie and Caubet, C{\'e}cile and Bascands, Jean-Loup and others,
Journal: PLoS One (2015): e0131416

References

View all 32 references: Citation Explorer
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
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
Evaluation of annexin V and Calcein-AM as markers of mononuclear cell apoptosis during human immunodeficiency virus infection
Authors: Palma PF, Baggio GL, Spada C, Silva RD, Ferreira SI, Treitinger A.
Journal: Braz J Infect Dis (2008): 108
Measurement of annexin V uptake and lactadherin labeling for the quantification of apoptosis in adherent Tca8113 and ACC-2 cells
Authors: Hu T, Shi J, Jiao X, Zhou J, Yin X.
Journal: Braz J Med Biol Res (2008): 750
Detection of apoptosis induced by new type gosling viral enteritis virus in vitro through fluorescein annexin V-FITC/PI double labeling
Authors: Chen S, Cheng AC, Wang MS, Peng X.
Journal: World J Gastroenterol (2008): 2174
Page updated on October 4, 2024

Ordering information

Price
Unit size
Catalog Number22830
Quantity
Add to cart

Additional ordering information

Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
InternationalSee distributors
Bulk requestInquire
Custom sizeInquire
Technical SupportContact us
Purchase orderSend to sales@aatbio.com
ShippingStandard overnight for United States, inquire for international
Request quotation

Spectral properties

Absorbance (nm)

527

Correction Factor (260 nm)

0.474

Correction Factor (280 nm)

0.306

Extinction coefficient (cm -1 M -1)

900001

Excitation (nm)

527

Emission (nm)

550

Quantum yield

0.311

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

Platform

Flow cytometer

Excitation405 nm laser
Emission525, 50 nm filter
Instrument specification(s)Pacific Orange channel

Fluorescence microscope

ExcitationViolet filter set
EmissionViolet filter set
Recommended plateBlack wall, clear bottom

Components

The detection of binding activity of Annexin V-mFluor™ Violet 550 and phosphatidylserine in Jurkat cells. Jurkat cells were treated without (Blue) or with 1 µM staurosporine (Red) in a 37 °C, 5% CO2 incubator for 5 hours, and then dye loaded with Annexin V-mFluor™ Violet 550 for 30 minutes. The fluorescence intensity of Annexin V-mFluor™ Violet 550 was measured with a FACSCalibur (Becton Dickinson) flow cytometer using 405 nm laser at Ex/Em = 405/550 nm.
The detection of binding activity of Annexin V-mFluor™ Violet 550 and phosphatidylserine in Jurkat cells. Jurkat cells were treated without (Blue) or with 1 µM staurosporine (Red) in a 37 °C, 5% CO2 incubator for 5 hours, and then dye loaded with Annexin V-mFluor™ Violet 550 for 30 minutes. The fluorescence intensity of Annexin V-mFluor™ Violet 550 was measured with a FACSCalibur (Becton Dickinson) flow cytometer using 405 nm laser at Ex/Em = 405/550 nm.
The detection of binding activity of Annexin V-mFluor™ Violet 550 and phosphatidylserine in Jurkat cells. Jurkat cells were treated without (Blue) or with 1 µM staurosporine (Red) in a 37 °C, 5% CO2 incubator for 5 hours, and then dye loaded with Annexin V-mFluor™ Violet 550 for 30 minutes. The fluorescence intensity of Annexin V-mFluor™ Violet 550 was measured with a FACSCalibur (Becton Dickinson) flow cytometer using 405 nm laser at Ex/Em = 405/550 nm.