Fig. 6. Rapid formation of peptide-MHC class II complexes derived from cytosolically delivered protein in endosomal compartments and their subsequent appearance on the cell surface of APCs. (A,B) Macrophages from C57Bl/6 mice were pulsed either exogenously in isotonic medium (A) or cytosolically in hypertonic medium (B) for 10 minutes with 5 mg/ml GST-E protein. Pulsed protein was washed off and cells were chased for different times indicated, fixed-permeabilized and stained with the Y-Ae antibody to detect formation of peptide-MHC class II complexes. (C,D) BMC-2 cells incubated with 5 mg/ml GST-E_52-68-myc protein coupled to the Chariot^TM reagent for 15 minutes at 0°C, were washed, chased for 15 minutes at 37°C, fixed-permeabilized and co-stained for LAMP-1 and Y-Ae antibodies to detect the intracellular Y-Ae+ complexes formed at this time. Right image is an overlay of the stained images in C. (D) A time course of detection by flow cytometry of intracellular Y-Ae⁺ complexes formed in BMC-2 cells after pulsing by the indicated methods and chased for different lengths of time. Scale bars, 10 µm.