2.7. Membrane permeability assay

Cells were seeded at 10⁶/2 ml in 24 well plates and treated with the following amounts of viability dyes: 100 ng/ml μM SYTOX green, 8 μg/ml PI, 150 ng/ml 7- aminoactinomycin 7AAD; Beckman-Coulter or 2.4 μM doxorubicin alone or in combination with 8 μM Elacridar or 8 μM MK571. Concentrations of efflux pumps inhibitors were earlier adjusted to maximal non-toxic doses. Immediately after treatment with viability dyes and/or efflux pumps inhibitors, 100 μl cell suspension was mixed with 100 μl PBS buffer and fluorescence in green (SYTOX green) or red (PI, 7AAD, doxorubicin) channel was measured by flow cytometry. Prior to the measurements, acquisition settings were adjusted to place the unstained cell population in the first decade on the logarithmic X axis. Measurements were repeated every 30 min for 6 h, in parallel for all cell lines. Median fluorescence intensity rather than mean fluorescence intensity was used since it was less influenced by the dead cell population (<10%).