Cd197

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.

Expanded spike-specific T cells and PBMCs collected five months post-vaccination were stimulated with the S1 peptide pools (1µg/ml, Oxford Immunotec) for 18 hours. Stimulated PBMCs and T cells were stained with fluorophore-conjugated monoclonal antibodies against CD3 (BD Biosciences), CD4 (BD Biosciences), CD8 (BD Biosciences), CD45RA (BD, Biosciences), CD69 (BD Biosciences), CD107 (BD Biosciences), CD134 (Thermo Fisher), CD137 (Thermo Fisher), and CD197 (BD Biosciences) for phenotypical characterization. All samples were acquired using a Fortessa flow cytometer (BD Biosciences) and the data was analyzed using FlowJo V10 software (BD Biosciences).