Abstract
This assay is used to count the number of cells that have undergone apoptosis. Apoptosis will be detected by initially staining the cells with Annexin V and propidium iodide solution followed by flow cytometry analysis. It is based on the principle that normal cells are hydrophobic in nature as they express phosphatidyl serine in the inner membrane (side facing the cytoplasm) and when the cells undergo apoptosis, the inner membrane flips to become the outer membrane, thus exposing phosphatidyl serine. The exposed phosphatidyl serine is detected by Annexin V, and propidium iodide stains the necrotic cells, which have leaky DNA content that help to differentiate the apoptotic and necrotic cells.
Keywords: Apoptosis, Annexin v, Propidium iodide, Necrotic cells
Materials and Reagents
Equipment
Procedure
Recipes
Acknowledgments
The authors thank Dr. Palanimuthu Ponnusamy Moorthy and Dr. Dhanya Haridas for critical reading of the apoptosis experimental protocol. We also thank Philip Hexley and Victoria Smith, Flow Cytometry Research Facility at UNMC, for their support.
References
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Does this number represent the initial amount of cells that have been seeded or for the ones that would be harvested?Yes. It represents the initial amount of cells that have been seededSecond question; how will the percentages be affected if I use less amount of staining mixture?For us it has worked very well for the mentioned concentration, if you want to use less amount of staining solution, we would recommend seeding less number of cells.