Figure 4

Increase in autolysosomal (autophagic) vacuoles in CCDD spleen cells. a) and b) Flow cytometry of unstained spleen cells from CCDWT and CCDD chimaeras. a) Average FL1 signal from five unstained preparation of spleen cells for CCDWT and CCDD. The average of CCDD is significantly greater than the CCDWT (Student's t-test two tailed, p < 0.02). b) Left panels show forward and side scatter profiles, which are indistinguishable between the two experimental groups. Right panels show contour plots of FL1 and FL2 autofluorescence. Note the different shape of the CCDD plot, reflecting increased fluorescence in both FL1 and FL2 channels. c) Spleen cells from CCDWT (left) and CCDD (right) were adhered to poly L-lysine, and fixed as described. Images were collected by confocal laser microscopy. The top left panel shows nuclear staining with DAPI, acquired using the UV laser. The top right panel shows images acquired using the Argon laser (excitation 488) and the emission settings for FITC fluorescence. The bottom left panel shows images acquired using the Argon laser and the emission settings for PE fluorescence. The bottom right shows a merged image of the three other panels. Scale bar – 10 μM. d) High power view of cells from CCDWT and CCDD. Scale bar – 10 μM. e) As for d, but arrow shows T cell stained with anti-CD3 FITC, containing autofluorescent granule. f) As for d, but arrow shows macrophage stained with anti-CD11b APC, containing autofluorescent granules.