Cell Meter™ Annexin V Binding Apoptosis Assay Kit Green Fluorescence Optimized for Flow Cytometry

The detection of binding activity of Annexin V-iFluor® 488 and phosphatidylserine in Jurkat cells. Jurkat cells were treated without (Blue) or with 20 µM camptothecin (Red) in a 37 °C, 5% CO2 incubator for 4-5 hours, and then dye loaded with Annexin V-iFluor® 488 for 30 minutes. The fluorescence intensity of Annexin V-iFluor® 488 was measured with a FACSCalibur (Becton Dickinson) flow cytometer using the FL1 channel.

Effect of FSS on apoptosis and necrosis in tubular cells. Confluent monolayers of HK-2 cells were submitted to FSS 0 (static) or FSS 0.5 Pa (FSS 0.5) for 48h. A/ Cells were stained with Annexin-V and then immediately subjected to analysis of phosphatidylserine externalization (Annexin-V fluorescence, X-axis) and Propidium Iodure (PI) uptake (PI fluorescence, Y-axis) using flow cytometry. Living, early apoptotic or necrotic (primary or secondary) cells were distinguished by the criteria of Annexin-V−/PI−(bottom left quadrant), Annexin-V+/PI− (bottom right quadrant) and Annexin-V+/PI+ (upper right quadrant), respectively. B/ Proportions of early apoptosis and necrosis cells were quantified and results are expressed as a percentage of the total population of cells. Data represent mean ± SEM of 7 experiments. *HK-2 cells were assessed for apoptosis and necrosis using Cell Meter Annexin V Binding Apoptosis Assay Kit (AAT Bioquest), according to the manufacturer's instructions. Source: Graph from <strong>Shear Stress-Induced Alteration of Epithelial Organization in Human Renal Tubular Cells</strong> by Damien Maggiorani, et al., <em>PLoS ONE</em>, July 2015.

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Telephone	1-800-990-8053
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Spectral properties

Correction Factor (260 nm)	0.21
Correction Factor (280 nm)	0.11
Extinction coefficient (cm ^-1 M ^-1)	75000¹
Excitation (nm)	491
Emission (nm)	516
Quantum yield	0.9¹

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
UNSPSC	12352200

Alternative formats

Cell Meter™ Annexin V Binding Apoptosis Assay Kit *Orange Fluorescence Optimized for Flow Cytometry*

Cell Meter™ Annexin V Binding Apoptosis Assay Kit *Red Fluorescence Optimized for Flow Cytometry*

Cell Meter™ Annexin V Binding Apoptosis Assay Kit *Deep Red Fluorescence Optimized for Flow Cytometry*

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

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

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

Overview SDS Protocol

Correction Factor (260 nm)

0.21

Correction Factor (280 nm)

0.11

Extinction coefficient (cm ^-1 M ^-1)

75000¹

Excitation (nm)

491

Emission (nm)

516

Quantum yield

0.9¹

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 FITC channel (green fluorescence).

Platform

Flow cytometer

Excitation	488 nm laser
Emission	530/30 nm filter
Instrument specification(s)	FITC channel

Components

Example protocol

AT A GLANCE

Protocol summary

Prepare cells with test compounds (200 µL/sample)
Add Annexin V-iFluor™ 488 stock solution
Incubate at room temperature for 30 - 60 mintues
Analyze cells using flow cytometer with 530/30 nm filter (FITC channel)

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

SAMPLE EXPERIMENTAL PROTOCOL

Treat cells with test compounds for a desired period of time (4 - 6 hours for Jurkat cells treated with camptothecin) 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.
Centrifuge the cells to get 1 - 5 × 10⁵ cells/tube.
Resuspend cells in 200 µL of Assay Buffer (Component B).
Add 2 µL of Annexin V-iFluor™ 488 (Component A) into the cells.
Optional: Add 2 µL of 100X Propidium Iodide (Component C) for necrosis cells.
Incubate at room temperature for 30 to 60 minutes, protected from light.
Optional: Add 200 to 300 µL of Assay Buffer (Component B) to increase volume before analyzing the cells with a flow cytometer.
Monitor the fluorescence intensity of Annexin V-iFluor™ 488 using a flow cytometer with 530/30 nm filter (FITC channel). Measure the cell viability using the 610/20 nm filter (PE-Texas Red channel) when propidium iodide is added into the cells.

Spectrum

Open in Advanced Spectrum Viewer

Spectral properties

Correction Factor (260 nm)	0.21
Correction Factor (280 nm)	0.11
Extinction coefficient (cm ^-1 M ^-1)	75000¹
Excitation (nm)	491
Emission (nm)	516
Quantum yield	0.9¹

Product Family

Name	Excitation (nm)	Emission (nm)	Extinction coefficient (cm ^-1 M ^-1)	Quantum yield	Correction Factor (260 nm)	Correction Factor (280 nm)
Cell Meter™ Annexin V Binding Apoptosis Assay Kit Orange Fluorescence Optimized for Flow Cytometry	557	570	100000¹	0.64¹	0.23	0.14
Cell Meter™ Annexin V Binding Apoptosis Assay Kit Red Fluorescence Optimized for Flow Cytometry	588	604	180000¹	0.53¹	0.05	0.04
Cell Meter™ Annexin V Binding Apoptosis Assay Kit Deep Red Fluorescence Optimized for Flow Cytometry	656	670	250000¹	0.25¹	0.03	0.03

Images

Figure 1. The detection of binding activity of Annexin V-iFluor® 488 and phosphatidylserine in Jurkat cells. Jurkat cells were treated without (Blue) or with 20 µM camptothecin (Red) in a 37 °C, 5% CO2 incubator for 4-5 hours, and then dye loaded with Annexin V-iFluor® 488 for 30 minutes. The fluorescence intensity of Annexin V-iFluor® 488 was measured with a FACSCalibur (Becton Dickinson) flow cytometer using the FL1 channel.

Figure 2. Effect of FSS on apoptosis and necrosis in tubular cells. Confluent monolayers of HK-2 cells were submitted to FSS 0 (static) or FSS 0.5 Pa (FSS 0.5) for 48h. A/ Cells were stained with Annexin-V and then immediately subjected to analysis of phosphatidylserine externalization (Annexin-V fluorescence, X-axis) and Propidium Iodure (PI) uptake (PI fluorescence, Y-axis) using flow cytometry. Living, early apoptotic or necrotic (primary or secondary) cells were distinguished by the criteria of Annexin-V−/PI−(bottom left quadrant), Annexin-V+/PI− (bottom right quadrant) and Annexin-V+/PI+ (upper right quadrant), respectively. B/ Proportions of early apoptosis and necrosis cells were quantified and results are expressed as a percentage of the total population of cells. Data represent mean ± SEM of 7 experiments. *HK-2 cells were assessed for apoptosis and necrosis using Cell Meter Annexin V Binding Apoptosis Assay Kit (AAT Bioquest), according to the manufacturer's instructions. Source: Graph from Shear Stress-Induced Alteration of Epithelial Organization in Human Renal Tubular Cells by Damien Maggiorani, et al., PLoS ONE, July 2015.

Citations

View all 11 citations: Citation Explorer

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

Phosphatidylserine exposure modulates adhesion GPCR BAI1 (ADGRB1) signaling activity
Authors: Lala, Trisha and Doan, Juleva K and Takatsu, Hiroyuki and Hartzell, H Criss and Shin, Hye-Won and Hall, Randy A
Journal: Journal of Biological Chemistry (2022): 102685

Atorvastatin attenuates pulmonary fibrosis in mice and human lung fibroblasts, by the regulation of myofibroblast differentiation and apoptosis
Authors: Yildirim, Merve and Kayalar, Ozgecan and Atahan, Ersan and Oztay, Fusun
Journal: Journal of Biochemical and Molecular Toxicology (2022): e23074

Itraconazole improves survival outcomes in patients with colon cancer by inducing autophagic cell death and inhibiting transketolase expression
Authors: Shen, Pei-Wen and Chou, Yu-Mei and Li, Chia-Ling and Liao, En-Chih and Huang, Hung-Sen and Yin, Chun-Hao and Chen, Chien-Liang and Yu, Sheng-Jie
Journal: Oncology letters (2021): 1--11

NGF-and BDNF-dependent DRG sensory neurons deploy distinct degenerative signaling mechanisms
Authors: de Le{\'o}n, Andr{\'e}s and Gibon, Julien and Barker, Philip A
Journal: Eneuro (2020)

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)

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

References

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Two-way trafficking of Annexin V positive cells between mother and fetus: determination of apoptosis at delivery
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Rhodamine B isothiocyanate doped silica-coated fluorescent nanoparticles (RBITC-DSFNPs)-based bioprobes conjugated to Annexin V for apoptosis detection and imaging
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Journal: Nanomedicine (2007): 266

Annexin A5-functionalized bimodal lipid-based contrast agents for the detection of apoptosis
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Analysis of cycloheximide-induced apoptosis in human leukocytes: Fluorescence microscopy using annexin V/propidium iodide versus acridin orange/ethidium bromide
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Quantum dots based probes conjugated to annexin V for photostable apoptosis detection and imaging
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