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.
Engbers-Buijtenhuijs
P
P
Department of Clinical Chemistry, Hospital Group Medisch Spectrum Twente, P.O. Box 50.000 7500 KA Enschede, The Netherlands.
Kamphuis
M
M
van der Sluijs Veer
G
G
Haanen
C
C
Poot
A A
AA
Feijen
J
J
Vermes
I
I
D017304:Annexin A5 / Q000494:pharmacology*; D023102:Anoikis; D017209:Apoptosis; D002448:Cell Adhesion; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D004396:Coloring Agents / Q000494:pharmacology*; D003470:Culture Media / Q000494:pharmacology; D053938:DNA Fragmentation; D004730:Endothelium, Vascular / Q000166:cytology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005063:Europium / Q000494:pharmacology*; D005434:Flow Cytometry; D006801:Humans; D008833:Microcirculation; D032389:Myocytes, Smooth Muscle / Q000166:cytology; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D014471:Umbilical Veins / Q000166:cytology
D017304:Annexin A5; D004396:Coloring Agents; D003470:Culture Media; D004791:Enzyme Inhibitors; D014409:Tumor Necrosis Factor-alpha; D005063:Europium; D014357:Trypsin