The Annexin V-FITC Apoptosis Detection kit is used to detect apoptotic cells by flow cytometry. Annexin V-FITC is a fluorescent probe which binds to phosphatidylserine in the presence of calcium. Apoptosis, or programmed cell death, is a mechanism of cells used to negatively select cells that are deleterious to the host. The cellular changes involved in the process include loss of phospholipid asymmetry during the early stages of apoptosis. In living cells, phosphatidylserine is transported to the inside of the lipid bilayer by the Mg-ATP dependent enzyme, aminophospholipid translocase.3 At the onset of apoptosis, phosphatidylserine, which is normally found on the internal part of the plasma membrane, becomes translocated to the external portion of the membrane. The phosphatidylserine becomes available to bind to the annexin V-FITC conjugate in the presence of calcium. The procedure consists of the binding of annexin VFITC to phosphatidylserine in the membrane of cells, which are beginning the apoptotic process, and the binding of propidium iodide to the cellular DNA in cells where the cell membrane has been totally compromised. Apoptosis may be either spontaneous or induced by incubating the cells with staurosporine. The cells are incubated with annexin V-FITC and propidium iodide. After a 10 minute incubation period at room temperature the cells are analyzed by flow cytometry. Annexin V-FITC is detected as a green fluorescence and propidium iodide is detected as a red fluorescence.
Annexin V FITC Conjugate, 500µl in 50 mM Tris-HCl, pH 7.5, containing 100 mM NaCl
Propidium Iodide Solution 2 ml: 1 mg/ml in 10 mM potassium phosphate buffer, pH 7.4, containing 150 mM NaCl
10X Binding Buffer 1 vial (20 ml): 100 mM HEPES/NaOH, pH 7.5 containing 1.4 M NaCl and 25 mM CaCl2
Flow Cytometry, Fluorescence Microscopy
20tests ; 50 tests ; 100 tests
Store between 2°C-8°C. Do not freeze.
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