A Novel Assay To Measure Loss Of Plasma Membrane Asymmetry During Apoptosis Of Adherent Cells In Culture.
Manon van Engeland, Frans C.S. Ramackers, Bert Schutte*, Chris P.M. Reutelingsperger1, Departments of Molecular Cell Biology & Genetics and Biochemistry1, University of Limburg, Maastricht, The Netherlands
Early during the process of apoptosis cells loose their membrane asymmetry while their membrane integrity remains. Based on this phenomenon we developed a staining procedure for adherent cell cultures using annexin V-binding to probe for phosphatidylserine exposure at the outer cytoplasmic membrane, in combination with propidium iodide staining, to verify the integrity of the membrane. The effect of cell harvesting on annexin V binding and membrane integrity was investigated. It appeared that trypsinisation or use of EDTA induced artificial annexin V binding to the cells, while labeling with annexin V in culture followed by harvesting using a rubber policeman allowed the discrimination of vital, apoptotic and damaged cells. Furthermore it is shown that annexin V labeling followed by brief fixation in cold methanol allows multiparameter flow cytometric analysis of apoptotic cells. An example is shown of a culture of lung cancer cells analysed 8 hours after induction of apoptosis by olomoucine. It appears that the annexin V-positive fraction is characterised by both loss of DNA and cytokeratin staining. Annexin V-positive cells appear as a characteristic sub G1 peak in the DNA histogram.