Figure 3

Flow cytometry analysis of P-gp and efflux activity transfers between MCF-7 and MCF-7/Doxo variants of the human breast cancer cell line. A-B, A 50:50 MCF 7:MCF 7/Doxo cell mixture was seeded on cultures dishes at day 0 and co-cultured. P-gp expression was immunodected by using a PE-conjugated UIC2 monoclonal antibody (A) and P-gp activity was followed with calceinAM as a fluorescent probe (B) after 0, 3, 4, 5 and 6 days of co-culture. In both cases (A & B) a sample of 10 000 cells was analyzed. In order to reduce the stochastic fluctuations, normalized binned logarithmic histograms were built from all-events list-mode raw data (see methods). In A, the peak (on the left side) of MCF-7 expressing low levels of P-gp corresponds to the sensitive cells. The peak on the right side corresponds to MCF-7/Doxo resistant cells. In B, the peak (on the right side) corresponds to the low efflux activity of sensitive MCF-7 cells, and the peak on the right corresponds to the MCF-7/Doxo resistant cells. C-D, total mass (sum of collected fluorescence light of a sample) of P-gp (C) and efflux activity (D) in co-cultures have been computed through days 0, 3, 4, 5 and 6 for 10 000 events analyzed by flow cytometry.