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

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-alpha/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-alpha 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.