Annexin V is used as a probe to detect cells that have expressed phosphatidylserine (PS) on the cell surface, an event found in apoptosis as well as other forms of cell death. The Annexin V affinity assay typically uses a conjugate of annexin V and a fluorescent or enzymatic label, biotin or other tags, or a radioelement, in a suitable buffer (Annexin V binding to PS is Ca²⁺ dependent). The assay combines Annexin V staining of PS membrane events with the staining of the cell nucleus with PI or AAD-7 to distinguish living cells from dead cells. Annexin V apoptosis detection is based on the observation that soon after initiating apoptosis, cells translocate the membrane phosphatidylserine (PS) from the inner (cytoplasmic-facing) leaflet of the plasma membrane to the cell surface. Once on the cell surface, PS can be easily detected by staining with a fluorescent conjugate of Annexin V, a protein that has a high affinity for PS. Detection can be analyzed by flow cytometry or by fluorescence microscopy.

This kit contains:

• Annexin V (APC): 500 µl
• 7-AAD Viability Staining Solution: 500 µl
• Positive Control: 5 ml
• 10X Binding Buffer

Target	Annexin V
Reactivity	General (All species)
Conjugation	APC
Excitation/Emission	651/660
Laser Line	647
Form	Liquid
Storage	10X Binding Buffer: Store at 4 °C for short-term storage. For long-term storage, aliquot and store at -20 °C. All other reagents: Store undiluted at 4 °C. Avoid exposure to light. Do not freeze.
Directions for use	Staining Protocol Dilute the 10X Binding Buffer solution to 1X Working Binding Buffer solution with distilled water. Harvest cells (about 1 × 105 cells per test), then wash once with cold PBS. Remove the PBS from the cell pellet. Wash again with cold 1X Working Binding Buffer, then centrifuge at 300 × g for 10 min at room temperature. Remove the Binding Buffer from the cell pellet. Resuspend cells in cold 1X Working Binding Buffer to a concentration of 1 × 106 cells/ml. Add 100 µl of cells (1 × 105 cells) to each appropriate tube. Add 5 µl of Annexin V-FITC to the appropriate tubes. Gently vortex each tube and incubate for 10 minutes at room temperature in dark. Add 5 µl of PI or 7-AAD solution and incubate for 5 min at room temperature in dark. Wash cells once in PBS, then resuspend in PBS. Analyze by flow cytometry within 4 hours.
Availability	Shipped within 5-10 working days.
Note	This product is for research use only.

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