In vitro quantification of pigment production and transfer in 2D co-cultures and 3D skin organotypic

Here, we present a protocol to set up and study 2D keratinocyte-melanocyte co-cultures and 3D full-thickness human skin equivalents. We describe steps for culturing of keratinocyte and melanocyte lines and the establishment of both 2D and 3D co-cultures. The cultures are utilized to measure melanin content and investigate mechanisms driving melanin production and transfer, through flow cytometry and immunohistochemistry. Culture conditions are highly amendable to different conditions, and analysis is simple and objective-thus allowing for medium to high throughput. For complete details on the use and execution of this protocol, please refer to Ng et al. (2022).¹.