The study of skin pigmentation requires determining the rate of melanin production in melanocytes and quantifying the rate of melanosome transfer to keratinocytes. In this article, the authors describe a method to quantify melanosome transfer using immunofluorescence microscopy coupled with automated image analysis of in vitro human melanocytes and keratinocytes in co-culture. In this method, the number of melanin capped keratinocyte nuclei is quantified.
Melanins are naturally occurring pigments which determine the skin, hair, and eye color in humans. The process of melanin production is called melanogenesis, which occurs in highly specialized cells called melanocytes. Melanin production and distribution is the result of a complex relationship between melanocytes and the surrounding keratinocytes in the epidermis of the skin.
Due to the optical properties of melanin, it can absorb UVB light, protecting the nuclei from UVB radiation-induced DNA damage and thus reducing the risk of cancer. When studying skin pigmentation, it is important to determine how the rate of melanin transfer from melanocytes to keratinocytes is modulated under different experimental conditions.
This article describes an image-based screen, coupled with an automated image analysis pipeline, developed within the open-source software Cell-Profiler, to determine the percentage of melanin capped keratinocytes in a co-culture of melanocytes and keratinocytes. The described protocol involves staining the melanosomes, using a specific fluorescently labelled antibody to visualize the formation of melanin caps over keratinocyte nuclei. The protocol can be used to study the effect of different drugs or treatments on melanosome transfer and for the analysis of UVB-induced changes in melanin cap deposition using co-culture of human melanocytes and keratinocytes.
Figure 1. The final image obtained after image processing for UVB unexposed co-culture (left) and UVB exposed co-culture (right). The overexposed green objects are melanocytes, the blue staining represents the keratinocyte nuclei, and the white circles are the melanosomes forming the melanin cap. The nuclei with magenta outline are negative for the melanin cap, and the nuclei with yellow outline are positive for the melanin cap. Scale bar is 20 μm.
Figure 2. Quantification of the percentage of keratinocytes with a positive melanin cap in UVB-exposed and UVB-unexposed co-cultures. n ¼ 6, **p < 0.01
The full article can be accessed here.
1Skin Research Institute of Singapore, A*STAR, Singapore, Singapore. 2Skin Research Institute of Singapore, A*STAR, Singapore, Singapore. leah.vardy@sris.a-star.edu.sg. 3School of Biological Sciences, Nanyang Technological University, Singapore, Singapore. leah.vardy@sris.a-star.edu.sg.
