Lipofectamine rnaimax sirna transfection reagent

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.

Cells were detached using 0.05% trypsin-EDTA (Invitrogen) and resuspended in serum-free DMEM medium at the concentration of 10⁶ cells/ml. Firstly, Cy3-BORIS-MB or Cy3-RANDOM-MB (200 nM) was incubated at room temperature in presence of 1 µl/ml of Lipofectamine RNAiMAX siRNA transfection reagent (Invitrogen) using Opti-MEM medium. The Lipofectamine RNAiMAX reagent was used as delivery vehicle since in our conditions it gave less background compared to other reagents such as Streptolysin (data not shown). After 10 min, the transfection mix was added to the suspended cells and together incubated for 1 hour at 37°C. Hoechst 33342 (Invitrogen) was added at concentration of 5 µg/mL during the last 10 min of incubation. Then, cells were washed using Phosphate Buffered Saline (PBS, Invitrogen) and resuspended in PBS with 5 mM EDTA. Transfected cells were cytocentrifugated onto glass slide using a cytospin centrifuge and examined under fluorescent microscope (Axioplan2 Imaging, Zeiss). The fluorescence signal of Cy3-coniugated MB was analyzed using the red channel and Hoechst 33342 fluorescence emission was observed under blue channel.

MM-ECs (5x10⁶) were transiently transfected for 48h and 72h with 25 nM and 50 nM of siRNAs specific for RICTOR (target sequences: a) GGGAAUACAACUCCAAAUA; b) GCGAGCUGAUGUAGAAUUA; c) GAAGAUUUAUUGAGUCCUA; d) GACACAAGCACUUCGAUUA) and negative control scramble siRNAs (SMART-pool; Dharmacon RNA Technologies, Lafayette, CO, USA), or with the transfection reagent alone (Lipofectamine, RNAiMAX siRNA transfection reagent, Invitrogen Corp).