Cytation 3 mv

For the adherent SUM1315 cell line, cells were seeded in IbiTreat 8-well μ-slides (Ibidi^®, Gräfelfing, Germany) at a concentration of 50 × 10³ cells per well. Slides were maintained for 3 days at 37 °C in a humidified incubator in order to obtain homogeneous confluent culture. The suspension DU4475 cells were directly harvested from the culture flask. For both cell lines, cells were fixed with a 4% para-formaldehyde (PFA, Sigma, Darmstadt, Germany) solution before being incubated in a 1 μM fluorescent conjugate PBS solution for 1 h. Three successive washes of 20 minutes were then conducted in PBS before imaging with a Cytation™3 MV (BioTek^®, Winooski, VT, USA) fluorescent microscopy module (M = 40×, fluorescence filters = GFP, RFP or Cy5). Cells mean fluorescence intensity was calculated with Gen5 3.08 software (BioTek^®) for three independent experiments. For each condition, fluorescence intensity was measured from 10 different imaging fields. For P-gp silencing, SUM1315 cells were exposed 72 hours to specific ABCB1 siRNAs diluted at 50 nM in culture medium and lipofectamine solution (Ambion®, reference AM51331 assay ID #4123/ABCB1, sequences 5′ > 3′: GGAUAUUAGGACCAUAAAUtt (sense), AUUUAUGGUCCUAAUAUCCtg (Antisense)). Cells were then fixed with para-formaldehyde 4% solution and stained with fluorescent conjugates or specific antibodies as previously described.

SUM1315 cells were seeded in IbiTreat 8-well slides at a concentration of 50 × 10³ cells per well and maintained at 37 °C in a humidified incubator until the cell layer reached 60% of confluence. Cells were then fixed with a 4% PFA solution. Fluorescent conjugate staining was conducted as previously described before anti-P-gp immunostaining. After a 1 hour aspecific sites saturation step with 1% bovine serum albumin (Sigma, Darmstadt, Germany) PBS solution, cells were incubated with clone F4 anti-P-gp monoclonal mouse antibody (Invitrogen, Carlsbad, CA, USA, catalog # MA5-13854, diluted at 1/75) for one hour. Three successive washes were then carried out in PBS before incubation with secondary goat anti-mouse Alexa Fluor™ 647 nm antibody or anti-mouse Alexa Flour™ 488 nm (Invitrogen, Carlsbad, CA, USA, catalog #A21236 or #A11001 diluted at 1/800). Cells were finally imaged with Cytation™3 MV (BioTek^®, Winooski, VT, USA) fluorescent microscopy module (M = 40×, fluorescence filters = GFP, RFP, & Cy5). The co-localization study and determination of Pearson's correlation coefficients was performed using JaCoP2 plugin of ImageJ software (NIH, Bethesda, MD, USA). The mean of Fisher transforms corresponding to Pearson correlation coefficients obtained on 10 different microscope fields was used.