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

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Abstract
Background: Adherent cells undergo apoptosis when detached from their home ground, a process called anoikis (homelessness). Methods: We developed a new and sensitive method to analyse apoptosis and anoikis of adherent cell types using a time resolved fluorometric assay with Europium-labelled Annexin V. Anoikis was induced with tumor necrosis factor-α /cycloheximide and three cell fractions of the cell cultures were prepared and analysed. Fraction 1 consisted of adherent cells, analysed while growing on their support (without detachment by trypsinisation). Fraction 2 contained detached cells due to anoikis (floating cells) and fraction 3 contained apoptotic bodies. Both fractions 2 and 3 were present in the culture medium and were isolated by differential centrifugation. Results: TNF-α treatment of three different types of adherent cell cultures induced a significant increase of the amount of floating cells (anoikis) and apoptotic bodies compared to control cell cultures. Also in the adherent cell fractions a small amount of apoptosis was observed. Conclusions: The novel time resolved assay provides the ability to analyse the cell death cascade in adherent cell cultures of the same sample at the same time in a sensitive and reproducible way.