5 6 carboxy 2 7 dichlorofluorescein diacetate cd fda

For staining of cells of the hepatic cell layer the sealing foil of the biochip (serving as cell substrate) was carefully removed with a scalpel. For staining of cells from the vascular layer the suspended membrane was similarly removed. Cells were then fixed with 4% paraformaldehyde for 10 min at room temperature (RT). Staining was done with antibodies against: MRP-2, PECAM-1 (Cell Signaling, Leiden, The Netherlands), von Willebrand factor, VE-Cadherin (BD Biosciences), ApoB (Santa Cruz, Heidelberg, Germany), ZO-1 (Life Technologies, Karlsruhe, Germany), CYP3A4 (Merck-Millipore, Schwalbach, Germany) CD163 (Biolegend, United Kingdom), CD197 (BD BioScience), CD68 (Santa Cruz Biotechnology, Heidelberg, Germany) and secondary antibodies goat-anti-mouse-Cy3, goat-anti-rabbit-Cy5 (Dianova, Hamburg, Germany), goat-anti-rabbit-AlexaFluor488 and DAPI (Life Technologies). Samples were embedded into fluorescent mounting medium (Dako, Hamburg, Germany). MRP-2 activity analysis was performed by incubation of HepaRG cell layers in serum free Williams E medium (GIBCO) containing 5 μM 5(6)-Carboxy-2′,7′-dichlorofluorescein diacetate (CD-FDA) (Sigma-Aldrich) at 37 °C for 15 min. Subsequently, imaging was performed on an AXIO Observer Z1 fluorescence microscope with Apotome 2 extension (Carl Zeiss AG, Jena, Germany). Image analysis was done with ImageJ2 software.

3D cultures were incubated 30 min with 5(6)-Carboxy-2′,7′-dichlorofluorescein diacetate CDFDA (10 μM) (Sigma-Aldrich), washed twice with PBS and incubated in serum-free medium for 120 min. The fluorescence (Ex.: 470 nm, Em.: 529 nm) was assessed using a fluorescence Eclipse Ni-E microscope (Nikon) equipped with a photonic camera Orca R2 (Hamamatsu).