MELANOSOME TRANSFER EVALUATION 199 until they reached confl uence. Human normal adult keratinocytes were separately seeded in Transwell® inserts at 40000 cells per insert, 24 hours before the beginning of the experiments. For the study of the Pmel 17 production, Transwell® inserts containing the keratinocytes were transferred into the multiwell culture plates containing the melanocytes. The co- cultures were then incubated for a 72-hour period in the absence (control), or in the pres- ence of niacinamide at 5 mM or of an Alaria esculenta extract at 0.01% (v/v). At the end of the incubation period, the Pmel 17 was quantifi ed in culture media and the monolay- ers were rinsed with PBS before the quantifi cation of the cell lysates total protein content. Pmel 17 ELISA ASSAY Pmel 17 ELISA development. Multiwell plates specially designed to perform ELISA assays were incubated overnight at 4°C with 100 μl of serial dilutions (0, 1, 5, 10, 50, 100, 500, and 1000 ng/ml) of the standard peptide. In order to test different primary and secondary antibody dilutions, all the experimental conditions were performed in quadruplicate (n = 4). At the end of this incubation period, the wells were emptied and non-specifi c binding sites were saturated by the addition of 300 μl per well of a 1% BSA solution in PBS. After an incubation period of one hour at room temperature, the wells were emptied and fi lled with 100 μl of a 1% BSA solution in PBS containing the anti-human Pmel 17 an- tibody tested at two different concentrations (0.3 and 1 μg/ml). After an incubation pe- riod of two hours at room temperature, the wells were washed three times with PBS containing 0.1% of Tween 20. The wells were then fi lled with 100 μl of a 1% BSA solu- tion in PBS containing the secondary antibody (two different concentrations were tested: 1/1000 and 1/10000) coupled to a peroxidase, and were incubated at room temperature for a two-hour period. At the end of this incubation period, the wells were again washed three times with PBS containing 0.1% of Tween 20, and 100 μl of a solution containing a peroxidase substrate was added to each well. After 20 minutes, the peroxidase reaction was stopped by adding 50 μl of a 2N H2SO4 solution. The colorimetric signal was ana- lyzed (two wavelengths of reading: 450 and 550 nm) by using an appropriate spectropho- tometry plate reader (Victor V, Perkin Elmer, Waltham, MA). For calculations, the signal obtained at 550 nm was subtracted from those obtained at 450 nm. Pmel 17 ELISA assay in co-culture media. The multiwell plates specially designed to per- form ELISA assays were incubated overnight at 4°C with 100 μl of serial dilutions of the standard peptide or of the samples to be assayed. At the end of this incubation period, non-specifi c binding sites were saturated by the addition of 300 μl per well of a 1% BSA solution in PBS. After an incubation period of one hour at room temperature, the wells were emptied and fi lled with 100 μl of a 1% BSA solution in PBS containing the anti- human Pmel 17 antibody (antibody dilution: 1 μg/ml). After an incubation period of two hours at room temperature, the wells were washed three times with PBS containing 0.1% of Tween 20. The wells were then fi lled with 100 μl of a 1% BSA solution in PBS con- taining the secondary antibody (antibody dilution: 1/10000) coupled to a peroxidase, and were incubated at room temperature for a two-hour period. At the end of this incubation period, the wells were again washed three times with PBS containing 0.1% of Tween 20, and 100 μl of a solution containing a peroxidase substrate was added to each well. After

JOURNAL OF COSMETIC SCIENCE 200 20 minutes, the peroxidase reaction was stopped by adding 50 μl of a 2N H2SO4 solu- tion. The colorimetric signal was analyzed (two wavelengths of reading: 450 and 550 nm) by using an appropriate spectrophotometry plate reader (Victor V, Perkin Elmer, Waltham, MA). For calculations, the signal obtained at 550 nm was subtracted from those obtained at 450 nm. MELANIN ASSAY Melanin determination was performed in cell lysates by measuring their optical absor- bance at 405 nm. PROTEIN ASSAY A protein assay was performed in cell lysates following the method of Bradford (7). STATISTICS Data are expressed as means ± S.E. of experiments realized, at least, in triplicate (n = 3). The statistical signifi cance was assessed by Student’s t-tests (* p 0.05 ** p 0.01). RESULTS AND DISCUSSION Pmel 17 ELISA ASSAY DEVELOPMENT In the fi rst part of our study we developed a “classical” ELISA assay to quantify Pmel 17 (see Materials and Methods). In order to get reproducible and accurate results, we chose to test two dilutions for the primary and the secondary antibodies. As shown in Figure 1, in all the conditions tested, the ELISA assay developed permitted us to detect from 10 to 1000 ng/ml (near 150 pmol/l to 15 nmol/l) of Pmel 17. It can also be noted that the better experimental conditions implied a dilution of 1 μg/ml for the primary antibody and of 1/10000 for the secondary one. This specifi c (data not shown, available from the antibody provider) and sensitive ELISA assay appears to be a good tool with which to follow up our investigation concerning the possibility of assessing melanosome transfer by quantifying the Pmel 17 released in the extracellular medium of melanocyte–keratinocyte co-cultures. MELANOSOME TRANSFER ASSESSMENT To test the hypothesis that it could be possible to assess melanosome transfer by quantify- ing the soluble Pmel 17 released in the extracellular medium after proteolytic ectodo- main shedding (3,4), we used melanocyte–keratinocyte co-cultures treated or not treated with a well-known inhibitor of this transfer, i.e., niacinamide.