Annexin V is a member of a calcium and phospholipid binding family of proteins with vascular anticoagulant activity. Results from in vitro experiments indicate that it may play a role in the inhibition of blood coagulation by competing for phosphatidylserine (PS) binding sites with prothrombin. In healthy cells, PS is usually kept in the inner-leaflet (the cytosolic side) of the cell membrane. When a cell undergoes apoptosis, one of the earliest detectable indicators is the loss of membrane asymmetry. No longer restricted to the cytosolic part of the membrane, PS is translocated to the outer-leaflet and becomes exposed on the surface of the cell.
ImmunoChemistry Technologies’ Equine Annexin V-Fluorescein Apoptosis Assay Kit provides a proven method for quickly and easily distinguishing two populations of dying cells from viable cells using recombinant fluorescein-conjugated Equine Annexin V and Propidium Iodide (PI). Cells with intact cell membranes and surface-exposed PS, a prominent feature of apoptosis, will stain positive for Annexin V-Fluorescein. PI is included in the kit to identify dying, later stage apoptotic cells which have lost plasma membrane integrity. These cells will become dually labeled with Annexin V-Fluorescein and Propidium Iodide (green and red fluorescence). Live non-apoptotic cells with intact plasma membranes will exclude PI and will remain unstained by the Annexin V-Fluorescein probe, assuming no treatment or cell cycle-associated event temporarily exposes the normally internalized, negatively charged PS entity. The kit also includes a specially formulated, calcium-based binding buffer which is required for Annexin V binding to occur. Analyze results using a flow cytometer.
1. Prepare samples and controls.
2. Dilute 10X Binding Buffer 1:10 WITH DiH2O.
3. Wash cells twice in ice-cold culture medium or PBS and resuspend in ice-cold 1X Binding Buffer at 5 x 105 – 1 x 106 cells/mL.
4. Reconstitute Equine Annexin V-Fluorescein in 1 mL Annexin Reconstitution Buffer, 1X.
5. Dilute reconstituted Annexin V-Fluorescein 1:5 in PBS.
6. Add diluted Annexin V-Fluorescein to each sample at ~1:10.
7. Dilute PI 1:10 in PBS.
8. Add diluted PI to each sample at ~1:20.
9. Incubate 15 minutes on ice and protected from light.
10. Add 400 µL 1X Binding Buffer to each sample.
11. Analyze with a flow cytometer. Annexin V-Fluorescein excites at 494 nm and emits at 521 nm. PI excites at 536 nm and emits at 617 nm.
Jurkat cells were treated with a negative control or staurosporine, for 4 hours, washed, then stained with ICT’s Equine Annexin V-Fluorescein Assay Kit and analyzed with an Accuri C6 flow cytometer. Treatment with staurosporine resulted in Annexin V labeling of 97.6% of the experimental cell population (27.8% + 69.8%), whereas the negative control treatment resulted in Annexin V labeling of 17.4% of the cell population (10.3% + 7.1%). The proportion of dual-labeled cells (Annexin V-Fluorescein (+)/PI (+), UR) increased significantly as a result of the treatment (Mrs. Tracy Murphy, ICT, 227:19).
• Target: Apoptosis
• Excitation/Emission: Annexin V-Fluorescein – 494 nm/521 nm
PI – 536 nm/617 nm
• Method of Analysis: Flow cytometry
• Types of Samples: cell culture
• Storage: 2-8°C
• Shipping: Ships overnight (domestic), International Priority Shipping
• Equine Annexin V-Fluorescein, lyophilized, 1 vial, #6644
• Annexin Reconstitution Buffer, 1X, 1 mL, 1 vial, #6639
• Annexin Binding Buffer, 10X, 10 mL, 3 bottles, #6640
• Propidium Iodide, 250 µg/mL, 1 mL, 1 vial, #638