Cells were prepared as previously described [25 (link)]. Briefly, cells in suspension (1 x 106 cells/ml) were incubated at 37°C for 1.5 hour in serum-free DMEM medium with Cy3-BORIS MB (200 nM) and Hoechst 33342 (5 μg/mL) in presence of a Lipofectamine RNAiMAX siRNA transfection reagent (Invitrogen). The cells were washed, resuspended in PBS—5 mM EDTA and cytocentrifugated onto glass slide using a cytospin centrifuge and then examined under a fluorescent (Axioplan2 Imaging, Zeiss) or a confocal (LSM 710 Quasar, Zeiss) microscope.
For ABCG2 fluorescence staining, cells were prepared as described beyond. After cytospin, cells were fixed with ice-cold acetone for 8 min and stained at 4°C overnight with rabbit anti-human ABCG2 antibody (Sigma) used at 1:20 dilution in PBS. Slides were washed with PBS and incubated for 1 hour at room temperature with donkey anti-rabbit secondary antibody labeled with Alexa Fluor 488 (Sigma) used at 1:500 dilution in PBS. The slides were then examined under fluorescent microscope.
For ABCG2 fluorescence staining, cells were prepared as described beyond. After cytospin, cells were fixed with ice-cold acetone for 8 min and stained at 4°C overnight with rabbit anti-human ABCG2 antibody (Sigma) used at 1:20 dilution in PBS. Slides were washed with PBS and incubated for 1 hour at room temperature with donkey anti-rabbit secondary antibody labeled with Alexa Fluor 488 (Sigma) used at 1:500 dilution in PBS. The slides were then examined under fluorescent microscope.
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