If you want the intact melanosomes that is challenging. Look at Watabe H et al 2005. Melanin tends to stick to nucleic acids and some proteins. When trying to quantify then you end up with numbers that read low. I have found that most melanomas that are in culture contain less melanin per million cells than normal melanocytes.

Dear Tatjana, perhaps my reply will not help that much but perhaps you can find some clues in the follwowing:

J Endocrinol November 1, 2005 187 293-302 doi: 10.1677/joe.1.06275

( cf: http://joe.endocrinology-journals.org/content/187/2/293.long )

by

Jennifer D Spencer, Bhaven Chavan, Lee K Marles, Sobia Kauser, Hartmut Rokos and Karin U Schallreuter

From Section:

:

In order to get the necessary quantities for the experiments, melanosomes were purified from human melanoma cells (FM55, FM94) and from murine B16 melanoma cells using the method described by Seiji et al.(1961)*). Briefly, a cellular homogenate is processed by centrifugation to provide a suspension of the large molecular weight organelles (mitochondria and melanosomes). In the second step, these organelles are separated on a sucrose gradient by ultracentrifugation allowing isolation of purified melanosomes of all maturation stages. Purified melanosomes were lysed via a repeated freeze/thaw cycle to obtain the melanosomal extract. The purity of the final preparation was examined by electron microscopy.

*) Seiji M, Fitzpatrick TB & Birbeck MS 1961

The melanosome: a distinctive subcellular particle of mammalian melanocytes and the site of melanogenesis. Journal of Investigative Dermatology 36 243–252.

and a second, perhaps also interesting article (see also Methods section):

Cell Logist. 2011 Jan-Feb; 1(1): 12–20.

(cf: http://ukpmc.ac.uk/articles/PMC3109459/ )

doi: 10.4161/cl.1.1.13638PMCID: PMC3109459 © 2011 Landes Bioscience

< Involvement of pigment globules containing multiple melanosomes in the transfer of melanosomes from melanocytes to keratinocytes> by

Hideya Ando, Yoko Niki, Masaki Yoshida, Masaaki Ito, Kaoru Akiyama, Jin-Hwa Kim, Tae-Jin Yoon, Mary S Matsui, Daniel B Yarosh, and Masamitsu Ichihashi.

Hope, it helps at least somehow,

best wishes and regards,

Wolfgang MUSS, PhD, SALZBURG, Austria

Thank you Dennis and Wolfgang! It helps.

Dear Tatjana, dear Dennies,

just as a final information (you might have got meanwhile elswhere) I would like to point you to the Article of:

Hideya ANDO, Yoko NIKI, Masaaki ITO, Kaoru AKIYAMA, Mary S. MATSUI, Daniel B. YAROSH and Masamitsu ICHIHASHI

J Invest Dermatol. 2012 Apr;132(4):1222-9

( cf. http://www.ncbi.nlm.nih.gov/pubmed/22189785 ).

The materials and methods section contains excellent and outline of the procedures (viz.: "Cultures, normal human epidermal melanocytes (derived from darkly pigmented newborn foreskins, passage up to 4; Cascade Biologics, Portland, OR) were maintained in medium 254 (Cascade Biologics) supplemented with a commercial cocktail of growth factors (HMGS, consisting of 10 ng mlˉ1 phorbol 12-myristate 13- acetate (PMA), 3 ngmlˉ1 human recombinant basic fibroblast growth factor, 3 mgmlˉ1 heparin, 500 nM hydrocortisone, 5 mgmlˉ1 insulin, 5 mgmlˉ1 transferrin, 0.2% (v/v) bovine pituitary extract, and 0.5% (v/v) fetal bovine serum) and an antibiotic/antimycotic solution (A5955; Sigma-Aldrich, St Louis, MO) at 37 1C in a humidified atmosphere with 5% CO2. Normal human epidermal keratinocytes (derived from darkly pigmented newborn foreskins, passage 3; Cascade Biologics) were cultured in medium 154 (Cascade Biologics, 0.2mM Ca2þ) supplemented with a commercial cocktail of growth factors (HKGS, consisting of 0.2 ng mlˉ1 human recombinant EGF, 0.18 mgmlˉ1 hydrocortisone, 5 mgmlˉ1 insulin, 5 mgmlˉ1 transferrin, and 0.2% (v/v) bovine pituitary extract) and an antibiotic / antimycotic solution....., < Isolation of pigment globules from the melanocyte culture medium > etc....

Hoping this will be of interest despite reporting this 14 days later, if you need the pdf I can't be of help because it is 16 MB+ (server size exeeding 18 MB, around 22 MB in an e-mail).

Perhaps you can tell us how your work has been going on with your melanoma cells, best regards and wishes,

Wolfgang MUSS, Salzburg - Austria

Wolfgang

Thank you for mentioning the J. Invest. article. I haven't seen this one.

Thank you Wolfgang! It is a good article. I have it in file now.