Annexins are a family of calcium-dependent phospholipid-binding proteins that preferentially bind phosphatidylserine (PS). Under normal physiologic conditions, PS is predominantly located in the inner leaflet of the plasma membrane. Upon initiation of apoptosis, PS loses its asymmetric distribution across the phospholipid bilayer and is translocated to the extracellular membrane leaflet marking cells as targets of phagocytosis. Once on the outer surface of the membrane, PS can be detected by fluorescently labeled Annexin V in a calcium-dependent manner.
In early-stage apoptosis, the plasma membrane excludes viability dyes such as propidium iodide (PI), 7-AAD. These cells will stain with Annexin V but not a viability dye, thus distinguishing cells in early apoptosis. However, in late stage apoptosis, the cell membrane loses integrity thereby allowing Annexin V to also access PS in the interior of the cell. A viability dye can be used to resolve these late-stage apoptotic and necrotic cells (Annexin V, viability dye-positive) from the early-stage apoptotic cells (Annexin V positive, viability dye-negative).
This kit is suitable for the identification and enumeration of dead cells, such as apoptotic or necrotic cells, by flow cytometry.
Kit Contents
A1373480 | Component | 20 T | 50 T | 100 T | Storage | Quantity Per Test |
A1373480A | 10x Annexin V Binding Buffer | 5 mL | 10 mL | 20 mL | 2-8℃. | 200 μL per 0.5-1.0x 10⁵ cells. |
A1373480B | Annexin V-Biotin | 450 μL | 1.5 mL | 2.5 mL | 2-8℃. Store in the dark. | 20 μL per 0.5-1.0x 10⁵ cells. |
A1373480C | Propidium iodide Staining Solution (PI) | 40 μL | 10 μL | 200 μL | 2-8℃. Store in the dark. | 2 μL per 0.5-1.0x 10⁵ cells. |
A1373480D | Streptavidin protein (FITC) | 40 μL | 10 μL | 200 μL | 2-8℃, Store in the dark. | 2 μL per 0.5-1.0x 10⁵ cells. |
Note: The recommended number of cells to stain per test is 0.5-1.0x10^5 cells.
Instruction for use
1. Before use, dilute the 10x binding buffer with deionized water to 1x (1 mL of 10x buffer plus 9 mL of deionized water);
2. After pre-cooling PBS and washing the cells twice, resuspend them in 1x Annexin V binding buffer at a density of 0.5-1.0x10^6 /mL;
3. Add 20 μL of fluorescently labeled Annexin V-Biotin and 0.5 μL of PI dye to each 200 μL cell suspension;
4. Gently vortex the cells and incubate at room temperature in the dark for 10 minutes;
5. Centrifuge at 400g for 5 minutes, remove the supernatant;
6. Dilute Streptavidin protein (FITC) with 1x Annexin V binding buffer at a ratio of 1:100（ 2 μL FITC +198 μL 1x Annexin V binding buffer ）
7. Add 200 μL of the diluted Streptavidin protein (FITC) and gently resuspend the cells;
8. Incubate at 4°C in the dark for 30 minutes;
9. Centrifuge at 400g for 5 minutes to remove the Streptavidin protein (FITC);
10. Add 200 μL of the binding buffer and complete the instrument analysis within 1 hour.
Matters needing attention
1. Please try to avoid light when using to slow down the quenching of fluorescence.
2. Propidium Iodide Solution is toxigenic and mutagenic; handle with care.
3. Due to the calcium dependence of the Annexin V:PS interaction, it is critical to avoid buffers containing EDTA or other calcium chelators during Annexin V experiments.
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