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

✍ Scribed by Walter Siegrist; Alex N. Eberle

• Publisher: Elsevier Science
• Year: 1986
• Tongue: English
• Weight: 521 KB
• Volume: 159
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(86)90327-1

✦ Synopsis

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; aher 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 (u-MSH, [Nle4, D-Phe']-a-MSH, [3'-iodo-Ty?]-ol-MSH, adrenocorticotropin (ACTH)( l-24), and ACTH( l-39) showed that the peptides had identical intrinsic activities and that the relative potencies were similar to those obtained with a tyrosinase assay. The EC50 of OI-MSH was 27 PM, i.e., about five-to sevenfold lower than that in the assays for tyrosinase or intracellular melanin. Thus, the new assay represents the most sensitive melanoma cell assay for MSH available to date. o 1986 Academic PKSS, tnc.