In healthy cells, phosphatidylserine (PS) only distributes in the inner layer of the cell membrane lipid bilayer. At the early stage of apoptosis, PS is turned from the inside of the lipid membrane to the outside. Annexin V, a Ca2+ dependent phospholipid binding protein with a molecular weight of 35-36 kD, has a high affinity to PS and can be used to detect to detect the externalization of PS. Annexin V is recognized as one of the sensitive indicators of early apoptosis. Apoptosis can be detected by fluorescence microscopy or flow cytometry using Annexin V probe conjugated to FITC (a green fluorescent). This kit also includes propidium iodide (PI), a dye for nucleic acid that cannot penetrate the intact cell membrane of healthy or early apoptotic cells. PI can penetrate the membrane of late apoptotic and necrotic cells, and stain these cells with red fluorescence. Using both Annexin V and PI, this kit can used to distinguish cells at different apoptotic periods, i.e. live cells (Annexin V-/PI-), early apoptotic cells (Annexin V+/PI-), and late apoptotic/dead cells (Annexin V+/PI+).
|
Component |
A211-01 (50 rxn) |
A211-02 (100 rxn) |
|
Annexin V-FITC |
250 µl |
500 µl |
|
PI Staining Solution |
250 µl |
500 µl |
| 1× Binding Buffer | 25 ml | 2 × 25 ml |
Store all components at 4-8℃ and protect from light.
The Annexin V-FITC can be stored at -20℃ for longer use.
Fig. 1. Detection of TRAIL-induced apoptosis of Jurkat cells by flow cytometry (A) 0 ng/ml. (B) 0.5 ng/ml. (C) 1.5 ng/ml. (D) 4.5 ng/ml. (E) 13.5 ng/ml. (F) Jurkat cells were induced apoptosis for 3 hours by 13.5 ng/ml TRAIL and then treated with 10 μg unlabeled Annexin V for 10 min, followed by Annexin V-FITC/PI staining. Unlabeled Annexin V, competiting with Annexin V-FITC, was used to confirm the specificity of staining