FIGURE 3.

Endolysosomal tubules colocalize with LysoTracker and soluble Ag. A, BMDCs expressing CD63-mRFP1 were loaded with 50 nM LysoTracker Green DND-26 (green) for 30 min and then washed twice in PBS. The cells were stimulated with 100 ng/ml LPS, and cells with endolysosomal tubules were imaged. Tubular compartments can be visualized using both CD63-mRFP1 and LysoTracker as evidenced by the merged image and the magnification of these structures. B, The endolysosomal tubules showed dynamic movement with heterogeneous staining of both signals over time. Images were obtained every two seconds. The arrows indicate the dynamic movement and heterogeneity of the endolysosomal tubules. C, Using LysoTracker Red DND-99 (red) and BMDCs from class II MHC-eGFP DCs, the colocalization of class II MHC and LysoTracker in tubular endosomes is seen. BMDCs expressing CD63-mRFP1 were incubated with 50 nM of OVA-Alexa Fluor 647 for 30 min before the addition of 100 ng/ml LPS and imaged 2 h later. D, Cells with endolysosomal tubules showed colocalization of CD63 and OVA (merged images and magnified image). E, Serial images were obtained every 2 s to demonstrate the dynamic behavior of the endolysosomal tubules containing CD63 and OVA-Alexa 647. Arrows denote one representative endolysosomal tubule.