Calcium, leukocyte cell death and the use of annexin V: fatal encounters.
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.
Trahtemberg
Uriel
U
The Laboratory for Cellular and Molecular Immunology, Rheumatology Unit, Department of Medicine, Hadassah-Hebrew University Medical Center, P.O. Box 12000, Jerusalem, 91120, Israel.
Atallah
Mizhir
M
Krispin
Alon
A
Verbovetski
Inna
I
Mevorach
Dror
D
D017304:Annexin A5 / Q000378:metabolism*; D002021:Buffers; D002118:Calcium / Q000378:metabolism*; D016923:Cell Death / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival; D002478:Cells, Cultured; D003470:Culture Media / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000378:metabolism*; D006801:Humans; D007962:Leukocytes / Q000166:cytology / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology; D008928:Mitochondria / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D013194:Staining and Labeling; D013997:Time Factors
D017304:Annexin A5; D002021:Buffers; D003470:Culture Media; D004791:Enzyme Inhibitors; D010718:Phosphatidylserines; D002118:Calcium