Figure 3.

In vitro interaction between Ag85B aggregates and professional antigen presenting cells. (A) Uptake of aggregated Ag85B by RAW264.7 macrophages as revealed by FACS analysis. Representative gated histograms depicting cumulative uptake of (a) native Ag85B only (F1); (b) FITC labeled native Ag85B-IFA complex (F1-FITC); and (c) FITC labeled Ag85B aggregates (F2-FITC). (B) Bar diagram displaying percent uptake of aggregated Ag85B by RAW264.7 cells. Quantitative data corresponding to FACS mediated uptake analysis of amyloidal Ag85B and controls. Data represent the means ±SD from three independent experiments and normalized to background fluorescence recorded in unlabeled F1 sample. (C) Fluorescence microscopy depicting uptaken aggregated antigen by APCs. RAW264.7 macrophages were incubated for 6 h with aggregated Ag85B (F2) and then stained with ThT for amyloid labeling and subsequently imaged (nuclei stained blue, while ThT labeled antigen in green; 100× oil immersion lens was used). (D) Immuno-localization of aggregated Ag85B inside RAW264.7 macrophages. The protein aggregate-mediated cytosolic delivery of Ag85B was probed with monoclonal primary antibodies against native Ag85B and its subsequent detection with Alexa Fluor 488 tagged secondary antibody (nuclei stained blue and antigen in green, 100× oil immersion lens).