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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.
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Catalog Number22839
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
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ShippingStandard overnight for United States, inquire for international
Spectral properties
Correction Factor (280 nm)0.254
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

OverviewpdfSDSpdfProtocol


Correction Factor (280 nm)
0.254
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


Component A: Annexin V- FITC (100X stock solution)1 vial (200 µL/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 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)0.254
Extinction coefficient (cm -1 M -1)73000
Excitation (nm)491
Emission (nm)516
Quantum yield0.92

Citations


View all 4 citations: Citation Explorer
miR-133a silencing rescues glucocorticoid-induced bone loss by regulating the MAPK/ERK signaling pathway
Authors: Wang, Gang and Wang, Fengbin and Zhang, Lecheng and Yan, Chao and Zhang, Yuelei
Journal: Stem Cell Research \& Therapy (2021): 1--14
Preliminary in vitro comparison of 111 In and 131 I labeled nimotuzumabs
Authors: Liao, Zhonghui and Li, Feize and Tang, Yu and Liu, Weihao and Gao, Jing and Lan, Tu and Yang, Jijun and Liao, Jiali and Liu, Ning and Yang, Yuanyou
Journal: Journal of Radioanalytical and Nuclear Chemistry (2021): 527--537
TBRG4 Knockdown Suppresses Proliferation and Growth of Human Osteosarcoma Cell Lines MG63 Through PI3K/Akt Pathway
Authors: Huang, Fei and Liao, Faxue and Ma, Guangwen and Hu, Yong and Zhang, Chi and Xu, Pengfei and Xu, Tangbing and Chang, Jun
Journal: OncoTargets and therapy (2020): 7271
Combined Treatments of Magnetic Intra-Lysosomal Hyperthermia with Doxorubicin Promotes Synergistic Anti-Tumoral Activity.
Authors: El, D Hajj Diab and Clerc, P and Serhan, N and Fourmy, D and Gigoux, V
Journal: Nanomaterials (Basel, Switzerland) (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