Uptake and intracellular distribution of neutral red in cultured fibroblasts.

Neutral red (2-methyl-3-amino-7-dimethylamino-phenazine) is taken up by cultured fibroblasts through a non-saturable process and its concentration in the cells reaches several hundred times that in the medium. The dye stains consistently discrete cytoplasmic granules; their size appears related to the level of cellular accumulation of neutral red. By isopycnic centrifugation of cytoplasmic extracts in sucrose gradients, we could clearly evidence an association of neutral red with (1) the lysosomal enzymes cathepsin D and N-acetyl-β-glucosaminidase; it is thought that neutral red accumulates in lysosomes by proton trapping; (2) cell constituents equilibrating at a median density of 1.15 g/cm3; this second compartment, with a concentration power as large as lysosomes, becomes apparent only when neutral red is more than 25 μM in the culture fluid; it serves as a temporary storage site, and the dye is thereafter transferred to lysosomes. We suggest this second compartment to be the Krinom vesicles, i.e. large autophagic vacuoles induced by and containing neutral red. Finally, a small amount of intracellular neutral red could be associated with either secretory or endocytic vesicles.