Assessment of cell survival and proliferation avoiding anoikis

3.2.1 Annexin V binding

6M-4

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on polystylene with campto thecin

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The externalization of phosphatidylserine after the detachment of cells was examined by Annexin V binding. As shown in Fig. 1, 6M-4 cells were apparently anoikis resistant during the first 6h on both the MPC and polystylene matrix. A larger fraction of the parental MDCK cells on the MPC matrix presented double positive signals than those on the polystylene matrix. After a 6

hour-exposure to camptothecin, which is an inhibitor of the gyrase responsible for DNA replication, the 6M-4cells were still resistant to Annexin V binding compared to the MDCK cells, although overnight contact with the drug was completely lethal.

on polystylene with campto thecin

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3.2.2 Respiratory activity

The quantitative respiratory activity of cells was measured using a REDOX indicator, alarmarBlue™ during the 6 days. As shown Fig. 2, the 6M-4 cells were resistant to MEP in contrast to the sensitivity of the parental MDCK cells. An unexpected stimulation of oxidative activity was observed by the addition of MEP to the 6M-4.

3.2.3 3H-thymidine and BrdU

incorporation into 6M-4 cells

Cell proliferation was directly examined using precursors of DNA synthesis. Quantitative 3H-thymidine incorporation into the cells during the 6 days was measured. The adherent and suspension 6M-4 cells showed a similar incorporation pattern during the culture period. Stimulation by MEP was observed, as well as the result of the respiratory assay, as shown in Fig. 3. As shown in Fig. 4-B, BrdU was incorporated into the suspension 6M-4 cells as well as the adherent cells. Interestingly, synchronous BrdU incorporation was observed in adjacent cells in floating aggregates.

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