Quantitative method of measuring phosphatidylserine externalization during apoptosis using electron paramagnetic resonance spectroscopy and annexin-conjugated iron.
We present here the application of a novel assay that measures the absolute amount of phosphatidylserine (PS) externalized on the surface of cells. Although the assay is based on the same annexin binding principle as the fluorescent flow cytometry assay, we use paramagnetic iron as the ultimate reporter molecule, establishing a linear relationship between signal amplitude and amount of PS on the cell surface, allowing a quantitative assay of PS externalization over a wide dynamic range. The application of this technique, alone and in concert with the PS oxidation method presented in the previous chapter, will greatly aid in studying the mechanistic connection between lipid peroxidation and translocation events during apoptosis.
Fabisiak
James P
JP
Department of Environmental and Occupational Health, University of Pittsburgh, PA, USA.
Borisenko
Grigory G
GG
Kagan
Valerian E
VE
D017302:Annexins / Q000737:chemistry*; D017209:Apoptosis; D002462:Cell Membrane / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D018922:HL-60 Cells; D006801:Humans; D007501:Iron / Q000737:chemistry*; D019169:Jurkat Cells; D015227:Lipid Peroxidation; D010718:Phosphatidylserines / Q000032:analysis*
D017302:Annexins; D010718:Phosphatidylserines; D007501:Iron