#6592 Annexin V-FITC Early Apoptosis Detection Kit Protocol
A. Solutions and Reagents
Note: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
20X Phosphate Buffered Saline (PBS)
(#9808): To prepare 1L 1X PBS, add 50ml 20X
PBS to 950ml dH20 and mix.
10X Annexin V Binding Buffer: To prepare 10mL 1X Annexin-V Binding Buffer, add 1ml 10X Binding
Buffer to 9ml dH20 and mix. Keep on ice.
B. Test Procedure
Collect cells of interest by centrifugation.
Wash cells with ice-cold PBS or culture medium.
Resuspend cells at 105 or 106 cells/ml with 1x Annexin V Binding Buffer. Aliquot 96µl of
cell suspension in an assay tube.
Add 1 µl Annexin V-FITC Conjugate and 12.5 µl Propidium Iodide (PI) Solution to each 96µl cell suspension. Allow
cells to incubate 10 min on ice in the dark.
Dilute the cell suspension to a final volume of 250 µl/ assay with ice-cold, 1X Annexin V Binding Buffer.
posted June 2020
Protocol Id: 2044
The Annexin V-FITC Early Apoptosis Detection Kit enables researchers to identify early apoptotic cells within a cell population. Annexin V-FITC conjugated protein binds to cell surfaces expressing phosphatidylserine, an early apotosis marker. Cells stained with propidium iodide (PI), a non-cell-permeable DNA dye, indicate necrotic cells. Cells stained with both PI and annexin V-FITC demonstrate later stage apoptosis and early necrosis. This kit provides enough reagent to perform 100 assays, based on a 250 μl assay volume.
Annexin V belongs to the annexin family of proteins consisting of over 160 members. All annexin family members share the same charactersistic of Ca2+-dependent binding to negatively charged phospholipid surfaces (1). During early apoptosis, phosphatidylserine (PS) is translocated from the cytosolic side of the plasma membrane to the cellular surface. This translocation exposes PS to the extracellular environment with the plasma membrane left intact (2). Annexin V protein demonstrates high affinity, specificity, and sensitivity for PS and can be used as a marker of early apoptosis. In order to rule out "leaky" necrotic cells, annexin V must be used in tandem with reagents that determine the integrity of the cell membrane, such as propidium iodide (2).