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

The detection of binding activity of Annexin V-mFluor™ Violet 500 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 500 for 30 minutes. The fluorescence intensity of Annexin V-mFluor™ Violet 500 was measured with a FACSCalibur (Becton Dickinson) flow cytometer using 405 nm laser at Ex/Em = 405/500 nm.
The detection of binding activity of Annexin V-mFluor™ Violet 500 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 500 for 30 minutes. The fluorescence intensity of Annexin V-mFluor™ Violet 500 was measured with a FACSCalibur (Becton Dickinson) flow cytometer using 405 nm laser at Ex/Em = 405/500 nm.
Ordering information
Price ()
Catalog Number22829
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
Correction Factor (260 nm)0.769
Correction Factor (280 nm)0.365
Extinction coefficient (cm -1 M -1)250001
Excitation (nm)410
Emission (nm)501
Quantum yield0.811
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Correction Factor (260 nm)
0.769
Correction Factor (280 nm)
0.365
Extinction coefficient (cm -1 M -1)
250001
Excitation (nm)
410
Emission (nm)
501
Quantum yield
0.811
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 405 laser with 525/40 emission filter set.

Platform


Flow cytometer

Excitation405 nm laser
Emission525/40 nm filter
Instrument specification(s)AmCyan channel

Fluorescence microscope

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

Components


Component A: Annexin V-mFluor Violet™ 500 (100X stock solution)1 vial (200 µL)
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 Annexin V-mFluor Violet™ 500 assay solution
  3. Incubate at room temperature for 30 - 60 minutes
  4. Analyze cells using flow cytometer with 525/40 nm filter (AmCyan 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™ 500:

  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™ 500 (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 a fluorescence microscope.

  8. Monitor the fluorescence intensity using a flow cytometer with 525/40 nm filter (AmCyan channel) or a fluorescence microscope with Violet filter set.

Analyze by using a flow cytometer:

  1. Quantify Annexin V-mFluor Violet™ 500 binding using a flow cytometer with 525/40 nm filter (AmCyan 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™ 500, 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™ 500 and visualized under a microscope.

  3. Analyze the apoptotic cells with Annexin V-mFluor Violet™ 500 under a fluorescence microscope using Violet filter set. Measure the cell viability using TRITC channel when propidium iodide is added into the cells. The green staining on the plasma membrane indicates the Annexin V-mFluor Violet™ 500 binding to PS on cell surface.

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Correction Factor (260 nm)0.769
Correction Factor (280 nm)0.365
Extinction coefficient (cm -1 M -1)250001
Excitation (nm)410
Emission (nm)501
Quantum yield0.811

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

Citations


View all 5 citations: Citation Explorer
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-Sébastien and Casemayou, Audrey and Ducasse, Laure and Grès, S and ra , undefined and Bellière, Julie and Caubet, Cécile and Basc, undefined and s, Jean-Loup and others, undefined
Journal: PloS one (2015): e0131416
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
Targeting a G-protein-coupled receptor overexpressed in endocrine tumors by magnetic nanoparticles to induce cell death
Authors: Sanchez, Claire and El Hajj Diab, Darine and Connord, Vincent and Clerc, Pascal and Meunier, Etienne and Pipy, Bernard and Payré, Bruno and Tan, Reasmey P and Gougeon, Michel and Carrey, Julian and others, undefined
Journal: Acs Nano (2014): 1350--1363

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
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
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
Two-way trafficking of Annexin V positive cells between mother and fetus: determination of apoptosis at delivery
Authors: Kolialexi A, Tsangaris GT, Anagnostopoulos A, Chondros D, Bagiokos V, Kitsiou S, Kanavakis E, Mavrou A.
Journal: Prenat Diagn (2007): 348
Rhodamine B isothiocyanate doped silica-coated fluorescent nanoparticles (RBITC-DSFNPs)-based bioprobes conjugated to Annexin V for apoptosis detection and imaging
Authors: Shi H, He X, Wang K, Yuan Y, Deng K, Chen J, Tan W.
Journal: Nanomedicine (2007): 266
Annexin A5-functionalized bimodal lipid-based contrast agents for the detection of apoptosis
Authors: van Tilborg GA, Mulder WJ, Deckers N, Storm G, Reutelingsperger CP, Strijkers GJ, Nicolay K.
Journal: Bioconjug Chem (2006): 741
Analysis of cycloheximide-induced apoptosis in human leukocytes: Fluorescence microscopy using annexin V/propidium iodide versus acridin orange/ethidium bromide
Authors: Baskic D, Popovic S, Ristic P, Arsenijevic NN.
Journal: Cell Biol Int (2006): 924
Quantum dots based probes conjugated to annexin V for photostable apoptosis detection and imaging
Authors: Le Gac S, Vermes I, van den Berg A.
Journal: Nano Lett (2006): 1863