Fluorochrome labeled ova

β-gal from K. phaffii was fluorochrome-labeled using the Alexa Fluor 647 Protein Labeling Kit (Thermofischer, Darmstadt, Germany). BM-DCs or HEK cells were incubated with 250 ng/ml fluorochrome-labeled OVA (Thermofischer, Darmstadt, Germany), 500 ng/ml fluorochrome-labeled β-gal or 500 ng/ml (unlabeled) GFP for 20 min. Cells were harvested and antigen uptake monitored by flow cytometry.

Armenian Hamster anti-CD80 (16-10A1), mouse anti-MHC I (28-14-8) and rat anti-CD40 (1C10) were from eBioscience (Frankfurt, Germany), rat anti-MR (MR5D3), mouse anti-His (AD1.1.10) and rat anti-CD86 (PO3) from AbD Serotec (Puchheim, Germany), rat anti-LAMP-1 (1D4B) from BD, rabbit anti-EEA1 (H-300) and all secondary antibodies from Santa Cruz (Heidelberg, Germany).
Fluorochrome-labeled OVA was from Thermo Scientific (Darmstadt, Germany), unlabeled OVA from Sigma (Taufkirchen, Germany), OVA_257-264 and OVA_323-339 from Tebu-bio (Offenbach, Germany), β-gal_497-504 (ICPMYARV) from PSL Peptide (Heidelberg, Germany). PNGase F and Endo H were obtained from New England Biolabs (Frankfurt, Germany).
Wildtype C57BL/6J mice, MR-deficient mice, OT I and OT II mice were bred under specific pathogen-free conditions and used in accordance with local animal experimentation guidelines. Mouse studies were approved by local regulatory agencies (Landesamt fuer Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen - LANUV NRW; Approval number §84-02.04.2012.A264). For all experiments, mice between 8 and 16 weeks of age were used. All mice were anaesthetized before euthanization by cervical dislocation.