A novel time resolved fluorometric assay of anoikis using Europium-labelled annexin V in cultured adherent cells

Adherent cells are dependent for survival from continuous engagement of cellular integrins to the extra cellular matrix. Detachment of adherent cells from the matrix induces almost immediately apoptosis, a phenomenon designated as ‘anoikis’ or homelessness (1). Activation of death receptors in adherent cells will cause detachment of those cells from their support inducing anoikis. Anoikis is of pertinent relevance to tissue homeostasis (2). Annexin V binding is extensively used to analyse apoptosis. During apoptosis (or necrosis) phosphatidyl serine is translocated from the inner to the outer leaflet of the plasma membrane and is recognised by Annexin V binding (3). We developed a new very sensitive method to analyse anoikis in adherent cell cultures using the principles of the Dissociation Enhanced Lanthanide Fluoro Immuno Assay (DELFIA®, Wallac Oy, Turku, Finland) (4). By using Europium-labelled Annexin V, the occurrence of apoptosis was measured in three different cell fractions derived from adherent cell cultures. The occurrence of apoptosis was analysed in cells which were still attached to the culture surface, anoikis was measured in detached cells (floating cells) and the final stage of the apoptotic pathway was investigated by analysing apoptotic bodies.