Culturing Cell Lines Expressing FcRn

The HEK293E cell line (ATCC) was cultured in RPMI-1640 GlutaMAX-containing medium (Sigma-Aldrich) supplemented with 10% fetal calf serum (FCS) 25 U/ml penicillin (Sigma-Aldrich) and 25 μg/ml streptomycin (Sigma-Aldrich). The MDCK cell line stably expressing hFcRn (MDCK-hFcRn) was generated by Drs. Jens Fischer and Alex Haas (Roche Pharma Research and Early Development), and cultured in cultured in DMEM with 15% FCS, 25 U/ml penicillin and 25 μg/ml streptomycin, as well as 100 μg/ml G418 (Sigma-Aldrich) to maintain expression of FcRn. HMEC-1 cells stably expressing HA-FcRn-EGFP (HMEC-1-FcRn^{57 (link)}) were cultured in MCDB131 medium (Gibco) supplemented with 2 mM L-glutamine, 25 μg/ml streptomycin, 25 U/ml penicillin, 10% FCS, 10 ng/ml mouse epidermal growth factor (BD Biosciences) and 1 μg/ml hydrocortisone (Sigma-Aldrich), and 100 μg/ml G418 and 50 μg/ml blasticidine (InvivoGen) to maintain FcRn expression. Percentage of HMEC-1 cells expressing HA-FcRn-EGFP in culture was above 85%, which was verified by visualizing EGFP-positive live cells, stained with the amine-reactive dye ViViD (Invitrogen), in combination with a biotinylated anti-FcRn mAb (ADM31^{95 (link)}) and allophycocyanin-conjugated streptavidin (PhycoLink) using a BD LSRFortessa.