|Storage and stability||
Stored at 2℃ - 8℃. Protected from prolonged exposure to light. Do not freeze !
All reagents are stable for one year from date of receipt under proper storage conditions.
|Safety Caution||7-AAD is a potential carcinogen. Wearing protective clothing, gloves, and eye/face protection is recommended in order to avoid contact with skin and eyes.|
The Annexin V-FITC/7-AAD Apoptosis Detection Kit has been tested on Jurkat cells treated with Camptothecin. Annexin V binding is calcium dependent and defined calcium and salt concentrations are required.
Investigators should note that the Annexin V-FITC/7-AAD Apoptosis Detection Kit has not been routinely tested on adherent cell types. During cell detachment and harvesting, membrane damage may occur, and then bring about a false positive result. If an adherent cell type was used, it is recommended to pre-test the cell dissociation method. And it is best to avoid using cell detach solution with EDTA.
Flow Cytometric Analysis of Annexin V-FITC staining. Jurkat cells were untreated (left panels), treated for 5 hours (middle panels) or 18 hours (right panels) with 4μM Camptothecin (Sigma,Cat.No C9911). Cells were incubated with Annexin V-FITC and 7-AAD and analyzed by flow cytometry. Untreated cells were primarily Annexin V-FITC and 7-AAD negative. After 5 hours treatment, there were primarily two populations of the cells: cells that were viable and not undergoing apoptosis (Annexin V-FITC and 7-AAD negative); cells undergoing apoptosis (Annexin V-FITC positive and 7-AAD negative). A minor population of cells were observed to be Annexin V-FITC and 7-AAD positive, they were in end stage apoptosis or already dead. After 18 hours treatment, the proportion of the cells at the end stage of apoptosis or dead ones (Annexin V-FITC and 7-AAD positive) was significantly increased.
♦Please quick-spin vial before opening, for maximal recovery of contents.
1. Wash cells twice with cold PBS, and then resuspend cells in 1×Binding Buffer at a concentration of 0.1×107~1×107 cells/mL.
1×Binding Buffer : Dilute 1 part of the 10×Binding Buffer to 9 parts of distilled water.
2. Transfer 100 μL of cell suspension to a tube.
3. Add 5 μL Annexin V-FITC and 5 μL 7-AAD, gently mix the cells and incubate for 15 min at room temperature (25℃) in the dark.
4. Add 400 μL of 1X Binding Buffer to each tube. Analyze by flow cytometry within 1 hr.
1. For accurate results, suggested the following controls to set up flow cytometry.
a) Negative control: Unstained cells.
b) Single color control: Cells stained with Annexin V-FITC (no 7-AAD). Cells stained with 7-AAD (no Annexin V-FITC).
The negative control and single color control are used to set up compensation and quadrants.
c) Experimental control: the untreated cell population, used to define the basal level of apoptotic and dead cells.
d) Optional control: To demonstrated the specific of Annexin V-FITC, cells incubated with purified recombinant Annexin V (10448-HNAE) to block Annexin V-FITC binding sites prior to adding Annexin V-FITC.
2. Do not use after expiration date.
3. Avoided to mix the different batches.
Apoptosis is a process of programmed cell death that occurs in multicellular organisms. It is a programmed cell death mechanism characterized by loss of plasma membrane asymmetry and attachment, cell shrinkage, nuclear fragmentation, chromatin condensation, chromosomal DNA fragmentation, and global mRNA decay. One of the earliest features is the change of plasma membrane.
Annexin V, also known as Annexin A5 (ANXA5), they are abundant intracellular proteins, and several different annexin gene products are expressed in all mammalian cells examined to date. Annexin V belongs to a family of Ca2+ binding proteins that undergo reversible Ca2+-dependent binding to phospholipids (PLs). In healthy cells, phosphatidylserine (PS) is predominantly located along the cytosolic side of the plasma membrane, upon initiation of apoptosis, the PS is translocated from the inner to the outer leaflet of the plasma membrane, thereby exposing PS to the external cellular environment, Annexin V has a high affinity for PS, and binds to cells with exposed PS. Annexin V may be conjugated to fluorochromes including FITC, PE. This format retains its high affinity for PS and thus serves as a sensitive probe for flow cytometric analysis of cells that are undergoing apoptosis.
7-AAD (7-amino-actinomycin D) has a high DNA binding constant and is efficiently excluded by intact cells, can be used in place of propidium iodide (PI) for the exclusion of nonviable cells. Different from PI, the 7-AAD has minimal spectral overlap with phycoerythrin (PE) and fluorescein isothiocyanate (FITC), and can be used in conjunction with PE and FITC-labelled antibodies in mulitcolor analysis.
In early stage apoptosis, the PS was exposed on the cell surface, but the plasma membrane excludes 7-AAD. These cells will stain with Annexin V but not 7-AAD, thus distinguishing cells in early apoptosis (Annexin V positive, 7-AAD negative). In late stage apoptosis, the cell membrane loses integrity thereby allowing 7-AAD access and bind to the DNA, at the same time allowing Annexin V access binding with the PS in the interior of the cell (Annexin V positive, 7-AAD positive). However this assay can't distinguish the cells that died undergone apoptotic with those that have died as a result of necrosis, in either case, the dead cells will stain with both Annexin V and 7-AAD.
1. Cederholm A. et al., 2007, Ann N Y Acad Sci. 1108: 96-103.
2. Schlaepfer DD. et al., 1992, Biochemistry. 311886-91.
3. Vermes I. et al., 1995, J Immunol Methods. 184 (1): 39–51.