Annexin V Kit

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Annexin V Assay Kit - 300 Tests thumbnail image 1

Annexin V Assay Kit - 300 Tests thumbnail image 2

Annexin V Assay Kit - 300 Tests thumbnail image 3

Annexin V Assay Kit - 300 Tests gallery image 1 — Jurkat cells were treated as shown with staurosporine at 1 μM to induce apoptosis. The cells were then stained with annexin V FITC (ANNEX300F) and ReadiDrop™ propidium iodide (1351101). Apoptotic cells positive for annexin V can be seen in the bottom right quadrant and dead cells positive for both annexin and PI in the top right quadrant. Healthy cells are negative for both stains. Data acquired on the ZE5™ Cell Analyzer.

Annexin V Assay Kit - 300 Tests gallery image 2 — Jurkat cells were treated as shown with staurosporine at 1 μM to induce apoptosis. The cells were then stained with annexin V FITC (ANNEX300F) and ReadiDrop™ propidium iodide (1351101). Apoptotic cells positive for Annexin V can be seen in the bottom right quadrant and dead cells positive for both Annexin and PI in the top right quadrant. Healthy cells are negative for both stains.

Annexin V Assay Kit - 300 Tests gallery image 3 — **Published customer image:**
Annexin V:FITC Assay Kit used for cell cycle analysis of cells treated with Arsenic trioxide and *Macuna macrocarpa* extract by flow cytometry.
**Image caption:**
Annexin V-FITC/propidium iodide (PI) analyses of arsenic trioxide (ATO)- and/or crude methanolic extract of *Mucuna macrocarpa* (CMEMM)-treated cells. HL-60 (a) and Jurkat (b) cells (1 × 10⁵ cells/mL) were first treated with 5 mM N-acetyl cysteine (NAC), 50 μM butylated hydroxytoluene (BHT), or 40 μM α-tocopherol (VitE) or untreated, followed by treatment with 0.1% DMSO (CTL), 2.5 μM ATO, and/or 50 μg/mL CMEMM as indicated for 24 h. Quantitative percentages of apoptotic cells of ATO/CMEMM-treated cells were measured by flow cytometry. Data represent the result from one of three independent experiments.

From: Lu KH, Lee HJ, Huang ML, Lai SC, Ho YL, Chang YS, Chi CW. Synergistic Apoptosis-Inducing Antileukemic Effects of Arsenic Trioxide and *Mucuna macrocarpa* Stem Extract in Human Leukemic Cells via a Reactive Oxygen Species-Dependent Mechanism.
Evid Based Complement Alternat Med. 2012;2012:921430.

Annexin V:Biotin Assay Kit

Annexin V:FITC Assay Kit

Product Type: Kits

Product Code	Applications	Datasheet	MSDS	Pack Size	List Price	Quantity
ANNEX300B	F			300 Tests
ANNEX300F	F			300 Tests

This test employs the property of Annexin V to bind to the membrane phospholipid phosphatidylserine (PS) in the presence of Ca²⁺. PS is exposed at the cell surface during the early stages of apoptosis. Detection of PS is a very sensitive method for detecting cells entering apoptosis, at a time point considerably ahead of nuclear changes such as DNA degradation.

The conjugation protocol used to prepare this product has not changed the native phospholipid binding properties of Annexin V. This protocol is designed to measure apoptosis easily and quickly in a sample of suspended cells.

View our complete list of formats and sizes of Annexin V kits

Product Details

Buffer Solution

Annexin V:FITC:
TRIS Buffered saline

Binding Buffer (after dilution):
10mM Hepes/NaOH
140mM NaCl
2.5mM CaCl₂

Preservative Stabilisers

0.02%	Sodium Azide (NaN₃)
1%	Bovine Serum Albumin

Preservative Stabilisers

Annexin V:FITC:
0.02% Sodium Azide (NaN₃)
1% Bovine Serum Albumin

Reagents in the Kit

Annexin V:Biotin	1 x 1.5ml vial
Propidium Iodide	2 x 1.6ml vial at 20ug/ml
Binding buffer	1 x 50ml vial at x 4 concentrate

Note: This assay also requires a streptavidin:FITC conjugate for visualisation (not supplied - see recommended useful reagents section).

Reagents in the Kit

Annexin V:FITC	1 x 1.5ml vial
Propidium Iodide	2 x 1.6ml vial at 20ug/ml
Binding buffer	1 x 50ml vial at x 4 concentrate

Storage Information

Storage: Store at +4^oC. DO NOT FREEZE.
This product should be stored undiluted. Should this product contain a precipitate we recommend microcentrifugation before use.
Storage: Store at +4^oC. DO NOT FREEZE.
This product should be stored undiluted. This product is photosensitive and should be protected from light. Should this product contain a precipitate we recommend microcentrifugation before use.
Shelf Life: 12 months from date of despatch.
Shelf Life: Please see label for expiry date.

More Information

UniProt: P08758 Related reagents
Entrez Gene: ANXA5 Related reagents
GO Terms: GO:0005509 calcium ion binding; GO:0007596 blood coagulation; GO:0004859 phospholipase inhibitor activity; GO:0007165 signal transduction; GO:0005544 calcium-dependent phospholipid binding; GO:0005737 cytoplasm; GO:0006916 anti-apoptosis; GO:0050819 negative regulation of coagulation
Regulatory: For research purposes only

Applications of Annexin V Kit

This product has been reported to work in the following applications. This information is derived from testing within our laboratories, peer-reviewed publications or personal communications from the originators. Please refer to references indicated for further information. For general protocol recommendations, please visit the antibody protocols page.

Application Name	Verified	Min Dilution	Max Dilution
Flow Cytometry			Neat
Flow Cytometry			Neat

Where this product has not been tested for use in a particular technique this does not necessarily exclude its use in such procedures. Suggested working dilutions are given as a guide only. It is recommended that the user titrates the product for use in their own system using appropriate negative/positive controls.

Instructions For Use: 1) Dilute the binding buffer 1:4 in distilled water (50ml binding buffer + 150ml distilled water).

2) Wash cells in PBS by gentle shaking or pipetting up and down.

3) Resuspend cells in 200ul pre-diluted binding buffer, adjusting to a cell density of 2-5 x 10⁵ cells/ml.

4) Add 5ul Annexin V:Biotin to 195ul of the cell suspension prepared in step 3.

5) Mix and incubate for 15 minutes at room temperature.

6) Wash cells twice in pre-diluted binding buffer.

7) Resuspend cells in pre-diluted binding buffer.

8) Add Streptavidin:FITC conjugate.

9) Mix and incubate for 30 minutes in the dark, at room temperature.

10) Wash cells in 200ul pre-diluted binding buffer.

11) Resuspend cells in 190ul pre-diluted binding buffer.

12) Add 10ul of the Propidium Iodide solution.

13) Analyse by flow cytometry.

The flow cytometer is preferably set such that the Mean Fluorescence Intensity of the Annexin V negative population is between 1 and 10. Optimal parameter settings can be found using a positive control. For a positive control, incubate the cells with 3% formaldehyde in buffer during 30 minutes on ice. Wash away the formaldehyde and suspend the cells in cold binding buffer at 2-5 x 10⁵ cells/ml. Proceed with step 2 as described above.
Instructions For Use: 1) Dilute binding buffer 1:4 in distilled water (50ml binding buffer + 150ml distilled water).

2) Wash cells in PBS by gentle shaking or by pipetting up and down.

3) Resuspend cells in 200ul of pre-diluted binding buffer, adjusting to a cell density of 2-5 x 10⁵ cells/ml.

4) Add 5ul Annexin V:FITC to 195ul of the cell suspension prepared in step 3.

5) Mix and incubate for 10 minutes in the dark, at room temperature.

6) Wash cells in 200ul of pre-diluted binding buffer.

7) Resuspend cells in 190ul pre-diluted binding buffer.

8) Add 10ul of the Propidium Iodide solution.

9) Analyse by flow cytometry.

The flow cytometer is preferably set such that the Mean Fluorescence Intensity of the Annexin V negative population is between 1 and 10. Optimal parameter settings can be found using a positive control. For a positive control, incubate the cells with 3% formaldehyde in buffer during 30 minutes on ice. Wash away the formaldehyde and suspend the cells in cold binding buffer at 2-5 x 10⁵ cells/ml. Proceed with step 2 as described above.

Useful Reagents Available

Description	Product Code	Pack Size	Applications
Streptavidin:FITC	710002	1 mg	F
Annexin V:Biotin Assay Kit	ANNEX100B	100 Tests	F
Annexin V:FITC Assay Kit	ANNEX100F	100 Tests	F
Annexin V:FITC Assay Kit	ANNEX300F	300 Tests	F
Streptavidin:FITC	STAR2B	1 mg	C F P
Annexin V:Biotin Assay Kit	ANNEX100B	100 Tests	F
Annexin V:FITC Assay Kit	ANNEX100F	100 Tests	F
Annexin V:Biotin Assay Kit	ANNEX20B	20 Tests	F
Annexin V:FITC Assay Kit	ANNEX20F	20 Tests	F
Annexin V:Biotin Assay Kit	ANNEX300B	300 Tests	F

Application Based External Images

Flow Cytometry

Product Specific References

References for Annexin V Kit

Lu, K.H. et al. (2010) In Vitro and In Vivo Apoptosis-Inducing Antileukemic Effects of Mucuna macrocarpa Stem Extract on HL-60 Human Leukemia Cells.
Integr Cancer Ther. 9: 298-308.
Koutsogiannaki S et al. (2015) Effects of cadmium and 17β-estradiol on Mytilus galloprovincialis redox status. Prooxidant-antioxidant balance (PAB) as a novel approach in biomonitoring of marine environments.
Mar Environ Res. 103: 80-8.
Yen, J. et al (2010) Glycine tomentella Hayata inhibits IL-1β and IL-6 production, inhibits MMP-9 activity, and enhances RAW264.7 macrophage clearance of apoptotic cells.
J. Biomed. Sci. 17: 83-91.
Chen, C.W. et al. (2010) The signals of FGFs on the neurogenesis of embryonic stem cells.
J Biomed Sci.17:33.
Lu KH et al. (2012) Synergistic Apoptosis-Inducing Antileukemic Effects of Arsenic Trioxide and Mucuna macrocarpa Stem Extract in Human Leukemic Cells via a Reactive Oxygen Species-Dependent Mechanism.
Evid Based Complement Alternat Med. 2012: 921430.
Smith, K. et al. (2011) Mono- and tri-cationic porphyrin-monoclonal antibody conjugates: photodynamic activity and mechanism of action.
Immunology. 132 (2): 256-65.
Koutsogiannaki S et al. (2015) Effects of cadmium and 17β-estradiol on Mytilus galloprovincialis redox status. Prooxidant-antioxidant balance (PAB) as a novel approach in biomonitoring of marine environments.
Mar Environ Res. 103: 80-8.

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