Calcium,leukocyte cell death and the use of annexin V: fatal encounters |
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Authors: | Uriel Trahtemberg Mizhir Atallah Alon Krispin Inna Verbovetski Dror Mevorach |
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Affiliation: | (1) The Laboratory for Cellular and Molecular Immunology, Rheumatology Unit, Department of Medicine, Hadassah-Hebrew University Medical Center, P.O. Box 12000, Jerusalem, 91120, Israel |
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Abstract: | One hallmark of programmed cell death (PCD) is redistribution of phosphatidylserine (PS) to the plasma membrane’s outer leaflet. Annexin V is widely used in cell death research due to its calcium-dependent ability to bind phosphatidylserine, thus marking apoptotic cells. However, calcium is invariably used at high concentrations in annexin V staining, at doses that can induce cell death. We used flow cytometric annexin V staining, together with propidium iodide and TMRM for determination of dissipation of mitochondrial potential, with a variety of calcium concentrations, cell media, and incubation times, to identify a possible bias in PCD determination of human primary leukocytes. Here we show that measurements of PCD in human monocytes, polymorphonuclear cells, and monocyte-derived dendritic cells using annexin V may be dramatically affected by calcium concentration, time of incubation on ice, and media choice. We propose a method that enables accurate and unbiased annexin V staining, without affecting results. Uriel Trahtemberg and Mizhir Atallah contributed equally to this publication. |
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Keywords: | Annexin V Apoptosis Calcium Culture medium Necrosis Programmed cell death |
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