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

The detection of binding activity of FITC-Annexin V to phosphatidylserine in Jurkat cells using Cell Meter™ FITC-Annexin V Binding Apoptosis Assay Kit. Jurkat cells were treated without (Blue) or with 1 μM staurosporine (Red) in a 37ºC, 5% CO2 incubator for 4-5 hours, and then dye loaded for 30 minutes. The fluorescence intensity of FITC-Annexin V was measured with a FACSCalibur (Becton Dickinson, San Jose, CA) flow cytometer using the FL1 channel.
The detection of binding activity of FITC-Annexin V to phosphatidylserine in Jurkat cells using Cell Meter™ FITC-Annexin V Binding Apoptosis Assay Kit. Jurkat cells were treated without (Blue) or with 1 μM staurosporine (Red) in a 37ºC, 5% CO2 incubator for 4-5 hours, and then dye loaded for 30 minutes. The fluorescence intensity of FITC-Annexin V was measured with a FACSCalibur (Becton Dickinson, San Jose, CA) flow cytometer using the FL1 channel.
The detection of binding activity of FITC-Annexin V to phosphatidylserine in Jurkat cells using Cell Meter™ FITC-Annexin V Binding Apoptosis Assay Kit. Jurkat cells were treated without (Blue) or with 1 μM staurosporine (Red) in a 37ºC, 5% CO2 incubator for 4-5 hours, and then dye loaded for 30 minutes. The fluorescence intensity of FITC-Annexin V was measured with a FACSCalibur (Becton Dickinson, San Jose, CA) flow cytometer using the FL1 channel.
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Spectral properties
Correction Factor (280 nm)0.35
Extinction coefficient (cm -1 M -1)73000
Excitation (nm)491
Emission (nm)516
Quantum yield0.92
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200
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OverviewpdfSDSpdfProtocol


Correction Factor (280 nm)
0.35
Extinction coefficient (cm -1 M -1)
73000
Excitation (nm)
491
Emission (nm)
516
Quantum yield
0.92
Our Cell Meter™ FITC-Annexin V binding assay 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 the green fluorescent FITC-Annexin V conjugate that specifically binds PS. The FITC-Annexin V conjugate has been demonstrated to selectively bind PS. This assay kit is optimized to monitor cell apoptosis using a flow cytometer with 488 nm laser and 530/30 nm emission filter (FITC channel).

Platform


Flow cytometer

Excitation488 nm laser
Emission530/30 nm filter
Instrument specification(s)FITC channel

Components


Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells with test compounds (200 µL/sample)
  2. Add Annexin V-FITC assay solution
  3. Incubate at room temperature for 30 - 60 minutes
  4. Analyze cells with a flow cytometer using FL1 channel (Ex/Em = 490/525 nm)

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

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 2 - 5 × 105 cells/tube.

  3. Resuspend cells in 200 µL of Assay Buffer (Component B).

  4. Add 2 µL of Annexin V-FITC (Component A) into the cells.

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

  6. Incubate at room temperature for 30 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 Annexin V-FITC using a flow cytometer with FL1 channel (Ex/Em = 490/525 nm). Measure the cell viability using FL2 channel when propidium iodide is added into the cells.

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Correction Factor (280 nm)0.35
Extinction coefficient (cm -1 M -1)73000
Excitation (nm)491
Emission (nm)516
Quantum yield0.92

Product Family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (280 nm)
Cell Meter™ APC-Annexin V Binding Apoptosis Assay Kit *Optimized for Flow Cytometry*651660730000-0.195
Cell Meter™ PE-Annexin V Binding Apoptosis Assay Kit *Optimized for Flow Cytometry*56557419600000.820.175

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References


View all 135 references: Citation Explorer
Dynamic analysis of apoptosis using cyanine SYTO probes: from classical to microfluidic cytometry
Authors: Wlodkowic D, Skommer J, Faley S, Darzynkiewicz Z, Cooper JM.
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Glycogen synthase kinase-3 and Omi/HtrA2 induce annexin A2 cleavage followed by cell cycle inhibition and apoptosis
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Journal: Mol Biol Cell (2009): 4153
Trivalent methylated arsenical-induced phosphatidylserine exposure and apoptosis in platelets may lead to increased thrombus formation
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Journal: Toxicol Appl Pharmacol (2009): 144
Eurycomanone induce apoptosis in HepG2 cells via up-regulation of p53
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Mobilization of lysosomal calcium regulates the externalization of phosphatidylserine during apoptosis
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Gold fluorescent annexin A5 as a novel apoptosis detection tool
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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)
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Peptidic targeting of phosphatidylserine for the MRI detection of apoptosis in atherosclerotic plaques
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