Bisbenzimide h

Live cell imaging analyses were performed by utilizing two different fluorescence microscope systems. Initial live cell imaging experiments as shown in Fig. 1 were performed on a confocal fluorescence microscopic system (Leica SP5, Leica Microsystems, Bensheim, Germany). Long term (48 hours) experiments as shown in Video S1 (see Supporting Information) were performed in an incubator with an inverted fluorescence microscope (Lumascope 500, Etaluma, Carlsbad, USA). Cells were co-cultured as described above. For confocal fluorescence microscopic experiments cells were plated on collagen type I (100 μg/ml; Invitrogen)-coated 100-mm tissue culture plates (Sarstedt, Nümbrecht, Germany) containing a 42-mm glass plate (H. Sauer Laborbedarf, Reutlingen, Germany). The nuclei of living cells were stained for 30 min with 5 mM bisbenzimide H (Sigma-Aldrich) at 37 °C in phenol red free Opti-MEM medium (Invitrogen). Glass plates with cells were implemented into a PeCon open chamber (PeCon, Erbach, Germany). Excitation wavelengths of 405 nm (bisbenzimide H), 481 nm (Pgp-EGFP), 561 nm (CMTPX, red) or 633 nm (eFluor670) were used. For 48 hours live cell imaging experiments, cells were co-cultured in an incubator with an inverted fluorescence microscope as described above. Images were taken every 10 minutes and excitation wavelengths of 488 nm were used.

pAECs grown on collagen type I-coated coverslips in six-well plates were washed with PBS 1X before fixation with 4% paraformaldehyde (Electron Microscopy Sciences, 15710; can be stored at −80 °C). After permeabilization in PBS 1X/FBS 10%/Triton 0.1% for more than 90 min, the primary antibody against RAGE (1/1000, ab37647, Abcam, Paris, France), HMGB1 (1/400, ab79823, Abcam, Paris, France), Myd88 (1/250, ab133739, Abcam, Paris, France), TIRAP (1/100, ab17218, Abcam, Paris, France), Diaphanous-1 (1/400, ab11173, Abcam, Paris, France), and p65 NF-κB (1/400, 8242, Cell signaling, Saint-Cyr-L’Ecole, France) was applied overnight at 4 °C. After three washes in the permeabilization buffer, the secondary antibody, anti-rabbit Alexa Fluor^® 488 (1/1000, A21206, Life Technologies, Villebon-Sur-Yvette, France), was incubated for 2 h at room temperature. Slides were washed three times in PBS 1X and incubated with Hoescht (15 min, dilution in PBS 1X 1/10,000; bisBenzimide H, 33258, Sigma-Aldrich, Saint-Quentin-Fallavier, France). Finally, slides were mounted with CitiFluor™ Tris-MWL 4-88 (Electron Microscopy Science, Hatfield, PA, USA) and examined under a Zeiss LSM800 Airyscan for cells. For the negative controls, incubation without the primary antibody was performed.

hCMEC/D3-MDR1-EGFP cells were plated on collagen type I (100 µg/ml; Invitrogen)-coated 100-mm tissue culture plates (Sarstedt) containing a 42-mm glass plate (H. Sauer Laborbedarf, Reutlingen, Germany). Four days after reaching confluence cells were pretreated with MMC (1 µM) for either 1 or 3 h in Opti-MEM (Invitrogen) at 37°C and 5% CO₂. Cells were stained for 0.5 h with 5 mM bisbenzimide H (Sigma-Aldrich) in Opti-MEM (Invitrogen) at 37°C and 5% CO₂. For confocal fluorescence microscopy glass plates with cells were fitted into a PeCon open chamber (PeCon, Erbach, Germany) and MMC (1 µM) in Opti-MEM without phenol red (Invitrogen) was added. Fluorescence images were taken every 3.6 min for the next hour using a Leica SP5 confocal fluorescence microscope with a 63× water objective (Leica Microsystems, Bensheim, Germany) in a climate box (PeCon) at 37°C. Excitation wavelengths of 405 nm (bisbenzimide H) or 481 nm (Pgp-EGFP) were used.