ABCB1 expression and functional analysis. (A) Membranous ABCB1 expression was scored across the DKFZ TMA and correlated with treatment risk group in 188 patients >3-17 years old. ABCB1 was significantly more likely (P ≤ 0.035) to be expressed in samples from high risk patients. (B) Relative expression of ABCB1 was calculated for each cell line using MED3 as a calibrator and GAPDH as a house keeping gene in the Pfaffl equation . ABCB1 is expressed in 6/8 MB cell lines. Data are expressed as the mean ± SEM of three experiments, each performed in triplicate. (C) Flow cytometry analysis of ABCB1 cell surface expression was carried out on MED1, MED3, DAOY and HEK-B1 control cell lines stained with anti-ABCB1 antibody (clone 4E3; unfilled histogram) or secondary antibody alone (filled histogram). Although only 5.8% of MED1 cells are positive for ABCB1 some cells expressed very high levels whereas MED3 showed no expression of ABCB1 (0.6 ± 0.4%). Representative dotplots can be found in Additional file 11: Figure S7. (D, E) ABCB1 function was tested by flow cytometry. Cells were incubated with either Rh123 alone (filled histogram) or in combination with VPL (D) or vardenafil (E) shown as unfilled histograms in each case. Inhibition of ABCB1 function was observed in 3.5% of MED1 cells consistent with the low% of cells expressing ABCB1. Similarly, there was no inhibition of function in MED3 cell line. Results in (C-E) represent mean ± SEM of three independent experiments.