Annexin V-FITC and PI in the Analysis of Apoptosis
Example from Research Core 1: Mechanisms of Toxicity and Cell Death. James P. Kehrer, Ph.D. & Vaidehee Desphande Mehta, University of Texas at Austin, College of Pharmacy, Division of Pharmacology and Toxicology.
The cell surface expression of phosphatidylserine translocated from the inner cytoplasmic membrane is considered an early apoptotic event as opposed to later evidence of apoptosis through morphological examination by microscopy which reveals chromatin condensation or membrane blebbing. In the above assay the Jurkat cell line is treated with etoposide, a topoisomerase 2 inhibitor (10mM, 18 Hr.s) as a positive (+) control and the experimental sample is treated with MK-886, an inhibitor of the 5-lipoxygenase activating protein, (40mM, 18 Hr.s). Four populations are resolved. The live cells are double negative and are seen in the lower left quadrant [A3]. Cells that are Annexin V-FITC(+)/PI(-) [A4] are apoptotic. The Annexin V-FITC(+)/PI(+) cell population [A2] has been described as 20 necrotic or advanced apoptotic. The last quadrant [A1] Annexin V-FITC(-)/PI(+) may be cells with stripped cytoplasmic membranes leaving isolated nuclei, cells in late necrosis, or cellular debris. It is recommended that each population be sorted for further examination by light microscopy techniques and DNA gel electropheresis to verify DNA degradation or morphological changes associated as benchmarks of apoptosis.
Annexin V-FITC/PI Protocol
Samples that need longer time for transport or any scheduling conflicts can be stored in Binding Buffer only. The Annexin V-FITC and PI can be added just prior to analysis.
DNA Content and Apoptosis 'TUNEL' Assay with anti-BrdU Cell Surface Labeling and 'TUNEL'
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Last updated: 11-March-02
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