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Ready-to-use
Annexin-V Apoptosis Detection Kit
Fast analysis of apoptosis in single cell suspensions
Background
MabTag’s Annexin V Apoptosis Detection Kit is designed to rapidly analyse different stages of apoptosis and cell death in single cell suspensions.
Annexin V binds specifically to phosphatidylserine (PS) which is located to the inner side of the cytoplasmic membrane in intact cells. But after initiation of apoptosis PS becomes rapidly translocated to the outer side of the cytoplasmic membrane where it is thought to play an important role in macrophage recognition, thus allowing the apoptotic cells to become rapidly phagocytosed.
The binding of annexin V to PS is Ca2+-dependent and therefore, a specific Ca2+-containing reaction buffer is needed during the binding process. At low PS concentrations a binding ratio of eight annexin V molecules to one PS has been reported making annexin V-conjugates ideal for identifying membrane changes associated with early apoptotic events.
In late apoptosis/necrosis cells loose their membrane integrity which allows further annexin V binding to PS located to the inner cell membrane. Therefore, MabTag’s Annexin V Apoptosis Detection Kit contains a viability dye such as propidium iodide (PI). This DNA-intercalating dye can only pass membranes which lost their integrity and therefore, allow the discrimination of early apoptotic cells (Annexin V+/PI-) from late apoptotic/necrotic cells (Annexin V+/PI+).
Cells were treated with UV for 30 minutes. After an incubation of 4 hours they were subsequently stained according to the procedure described below.
1. viable cells (double-negative)
2. early apoptotic cells (annexin V-positive/PI-negative)
3. late apoptotic/necrotic cells (double-positive)
Characteristics of Fluorochromes
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Fluorochrom
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Absorption peak (nm)
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Emission peak (nm)
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Excitation at (nm)
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FITC
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495
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519
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488
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APC
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650
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660
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633
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Propidium iodide
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536
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617
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488
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Reagents provided
500 µl Annexin V FITC / APC / Biotin conjugate; (with 1% BSA / 0.1% NaN3 as preservative)
500 µl propidium iodide [20 µg/ml]
10 ml Annexin V binding buffer (10x concentrate)
Staining procedure
1. Dilute an appropriate amount of 10x Annexin-V binding buffer with deionized or distilled water in 1:10 ratio
2. Wash cells with culture medium or PBS
3. Resuspend 104 – 106 cells in 90 µl of diluted (1x) Annexin-V binding buffer.
4. Add 5 µl of Annexin-V conjugate and 5 µl of propidium iodide solution.
5. Incubate 20 minutes in the dark.
6. Add 400 µl of Annexin V binding buffer (1x).
7. Centrifuge at 400 x g for 5 minutes.
8. Resuspend cells in approx. 500 µl Annexin-V binding buffer (1x) and analyze sample by flow cytometry or fluorescence microscopy.
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