Human skin-derived mast cells after GD3- and Kit-dependent immunoaffinity purification and coculture with fibroblasts.

Dispersed and density-dependent sedimentation-enriched skin-derived mast cells were purified using anti-GD3 mAb and were then labeled with PE-anti-Kit mAb. (A) A portion of these cells was placed on a slide, air dried, and stained with toluidine blue. Mast cells exhibit metachromatic purple cytoplasm and blue nuclei. (B) Another portion of these cells was subjected to sorting of individual cells onto fibroblast monolayers, which were then cultured with rhuSCF (100 ng/mL) for 3 weeks and stained with anti-tryptase mAb (naphthol AS-BI/new fuchsin). Positive cells stain red. Panel B shows a well containing multiple tryptase-positive cells. Original magnifications at the time of photomicroscopy were × 400 for panel A, and × 200 for panel B (inverted microscope).