A novel time resolved fluorometric assay of anoikis using Europium-labelled Annexin V in cultured adherent cells
Engbers-Buijtenhuijs, P. and Kamphuis, M. and Sluijs Veer, G. van der and Haanen, C. and Poot, A.A. and Feijen, J. and Vermes, I. (2005) A novel time resolved fluorometric assay of anoikis using Europium-labelled Annexin V in cultured adherent cells. Apoptosis, 10 (2). pp. 429-437. ISSN 1360-8185
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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-agr /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-agr 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.
|Copyright:||© 2005 Springer|
Science and Technology (TNW)
Electrical Engineering, Mathematics and Computer Science (EEMCS)
|Link to this item:||http://purl.utwente.nl/publications/71128|
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