Identification and quantification of aminophospholipid molecular species on the surface of apoptotic and activated cells

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Thomas, Christopher P. ; Clark, Stephen Robert ; Hammond, Victoria Jayne ; Aldrovandi, Maceler ; Collins, Peter William ; O'Donnell, Valerie Bridget (2014)

This protocol measures externalization of aminophospholipids (APLs) to the outside of the plasma membrane using mass spectrometry (MS). APL externalization occurs in numerous events, and it is relevant for transplant medicine, immunity and cancer. In this protocol, externalized APLs are chemically modified by using a cell-impermeable reagent (sulfo-NHS-biotin), and then they are isolated via a liquid:liquid extraction and quantified by reverse-phase liquid chromatography tandem MS (LC-MS/MS) against in-house-generated standards. This protocol describes a complementary method to existing assays that are not quantitative (e.g., annexin V flow cytometry), and it is applicable to the study of membrane reorganization in all cell types during apoptosis (e.g., during development, cancer, psychiatric disorders and other conditions, aging, vesiculation and cell division). The protocol takes ∼2–4 d, including the generation of standards.
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    Final analyte concentration 1 ng/μl 0.5 pg/μl 0.1 ng/μl 50 pg/μl 10 pg/μl 5 pg/μl 1 pg/μl 0.5 pg/μl 0.1 pg/μl

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