Conditioned Medium from Irradiated Keratinocytes as a Tool for Investigating Communication within the Melano‐epidermal Unit

Keratinocytes and melanocytes form the melano-epidermal unit, whose communication is essential for maintaining the integrity, homeostasis, and functionality of the skin. This interaction is mediated by keratinocytes' release of various soluble factors, which modulate key processes in melanocytes such as melanogenesis, proliferation, adhesion, and cellular differentiation. Exposure of keratinocytes to ultraviolet (UV) radiation can induce changes in their secretion profile, directly influencing the response of melanocytes and, consequently, skin pigmentation and protection against environmental aggressions. In this article, the presented protocol was developed and validated for obtaining conditioned medium from keratinocytes irradiated with UVA or UVB, followed by evaluation of cell viability as a criterion to determine the maximum applicable radiation dose. The conditioned medium obtained was applied to melanocyte cultures to investigate the effects mediated by secreted factors following radiation exposure. The methodology presented constitutes a tool for studying cellular communication under environmental stress conditions and may be used for developing photoprotective agents, for deepening understanding of keratinocyte-melanocyte interactions following UV radiation, and for contributing to the elucidation of mechanisms regulating pigmentary disorders. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Preparation, UV irradiation, and handling of conditioned medium from keratinocytes and application to melanocyte cultures.