In situ melanin assay for MSH using mouse B16 melanoma cells in culture

Abstract

A sensitive in situ melanin assay using cultured mouse B16 melanoma cells is described for structure-activity studies with melanocyte-stimulating hormone (MSH) peptides. B16 Cells were seeded at a density of 2500 cells per well in 96-well microtest tissue culture plates; after 24 h the cells were incubated in the presence of serial dilutions of MSH peptides for 3 to 5 days. The melanin released into the medium of each well was then determined spectrophotometrically at a wavelength of 405 nm using an automatic microplate reader calibrated against synthetic melanin. Studies with α-MSH, [Nle 4, d-Phe 7]-α-MSH, [3′-iodo-Tyr 2]-α-MSH, adrenocorticotropin (ACTH)(1–24), and ACTH(1–39) showed that the peptides had identical intrinsic activities and that the relative potencies were similar to those obtained with a tyrosinase assay. The EC 50 of α-MSH was 27 p m, i.e., about five- to sevenfold lower than that in the assays for tyrosinase intracellular melanin. Thus, the new assay represents the most sensitive melanoma cell assay for MSH available to date.