Culturing of Cell Lines for FcRn Studies

HEK293E cells (ATCC, Manassas, VA, USA) and the J558L murine myeloma cell line stably producing NIP-specific hIgG1-IHH were cultured in RPMI 1640 (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Sigma-Aldrich), 2 mM l-glutamine, 25 μg/ml streptomycin, and 25 U/ml penicillin (all from Bio Whittaker). The parental human microvascular endothelial cell line (WT HMEC1) and HMEC1 stably expressing HA-hFcRn-EGFP (HMEC1-hFcRn)^{38 (link)} were grown in MCDB 131 medium (Gibco) supplemented with 10% heat-inactivated FCS, 2 mM l-glutamine and 25 µg/ml streptomycin, and 25 U/ml penicillin, 10 ng/ml mouse epidermal growth factor (PeproTech) and 1 µg/ml hydrocortisone (Sigma-Aldrich). Medium for HMEC1-hFcRn was also supplemented with 5 µg/ml blasticidin (InvivoGen) and 100 µg/ml G418 (Sigma-Aldrich) to maintain stable expression of hFcRn. High five cells (Invitrogen) were grown in Express FIVE SEF medium (Invitrogen) supplemented with 18 mM l-glutamine and 1% antibiotic–antimyotic (Invitrogen). All cell lines were negative for mycoplasma contamination (MycoAlert^TM PLUS Mycoplasma detection kit, Lonza).