ANNEXIN V:FITC ASSAY KIT

Annexin V Kit allows fluorescent detection of annexin V bound to cells undergoing apoptosis. Apoptosis, or programmed cell death, is an essential process, by which organisms ensure that critically damaged or mutated cells are removed. This process is highly regulated by a complex interplay of proteins.

During the early stages of apoptosis, the plasma membrane of a cell is rearranged, resulting in changes to the cell membrane’s phospholipid asymmetry. Phosphatidylserine (PS), a phospholipid located within the cell membrane, is translocated and displayed on the outer plasma membrane (Fadeel and Xue, 2009). Annexin V; a protein encoded by the ANXA5 gene, has a high binding affinity for PS in the presence of Ca²⁺, and is therefore used as a tool for detecting early apoptotic cells.

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.

Storage Information

Store at +4°C. DO NOT FREEZE.
This product should be stored undiluted.

 

Applications

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

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

ANNEXINF

1) Dilute the 10x Binding Buffer to 1x in distilled water (1 ml 10x Binding Buffer + 9 ml distilled water).

2) Centrifuge and resuspend cells in PBS, adjusting to a cell density of 1 x 10⁶ cells/ml.

3) Transfer 100 μl cells to a 96-well flat bottom plate.

4) Centrifuge and resuspend cells in 95 μl 1x Binding Buffer.

5) Add 5 μl of FITC conjugated Annexin V

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

7) Centrifuge and resuspend cells in 200 μl of 1x Binding Buffer.

8) Resuspend cells in 200 μl of 1x Binding Buffer and add viability dye of choice at previously optimized concentration (e.g. DAPI or PI at 1 μg/ml).

9) Analyze by flow cytometry immediately. If not analyzed immediately, store at 2-8°C in the dark for maximum 4 hours, experimental design permitting.