title: A new spectrophotometric method for simple quantification of melanosomal transfer from melanocytes to keratinocytes.
abstract: Three major difficulties must be overcome to establish a quantitative method for melanosomal transfer analysis: (i) establishing a three-dimensional co-culture reassuring direct melanocyte to keratinocyte transfer, (ii) separation of melanocytes and keratinocytes following co-culture and (iii) melanosome quantification in each cell population. Melanocytes and keratinocytes are cultured on the opposite sides of the porous membrane of hanging cell inserts (1μm pores, 2×10(6) pores/cm(2) ). Cell separation is performed after 3days of co-culture by simple trypsinisation. Melanosome quantification in separated cell populations was accomplished by an ELISA-like method using gp-100 as the antigen. Melanocytes and keratinocytes come into 'direct' contact through the pores, and melanosomal transfer is accomplished without cell passage through the membrane. Cell separation by simple trypsinisation results in pure melanocyte and keratinocyte populations. Melanosome quantification by the ELISA-like method proved to be sensitive and specific to distinguish the known inhibitors and inducers of melanosomal transfer.
authors.affiliation: Department of Dermatology, Geneva University Hospital, Geneva, Switzerland. behroozkasraee@yahoo.com
authors.forename: Behrooz
authors.initials: B
authors.lastname: Kasraee
authors.forename: Marcela
authors.initials: M
authors.lastname: Pataky
authors.forename: Damjan S
authors.initials: DS
authors.lastname: Nikolic
authors.forename: Pierre
authors.initials: P
authors.lastname: Carraux
authors.forename: Vincent
authors.initials: V
authors.lastname: Piguet
authors.forename: Denis
authors.initials: D
authors.lastname: Salomon
authors.forename: Olivier
authors.initials: O
authors.lastname: Sorg
authors.forename: Jean-Hilaire
authors.initials: JH
authors.lastname: Saurat
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002469:Cell Separation; D018920:Coculture Techniques; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005434:Flow Cytometry; D015603:Keratinocytes / Q000648:ultrastructure*; D008544:Melanocytes / Q000648:ultrastructure*; D008546:Melanoma, Experimental / Q000648:ultrastructure; D020460:Melanosomes / Q000648:ultrastructure*; D051379:Mice; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D013053:Spectrophotometry / Q000379:methods*