Optimized melanocyte–keratinocyte co-culture for studying cell–cell interactions. (A) Dendritic branching of an individual melanocyte within an intact epidermal sheet, microinjected with Lucifer yellow. (B) Melanocytes in co-cultures have similar morphology to melanocytes in situ (A). Mosaic labeled melanocytes (iRFPmem, magenta) and keratinocyte (eGFPmem, green) in the bottom layer of mature co-culture. (C) Both K14-positive keratinocytes (basal layer) and keratinocytes with varying levels of K10 (top layer) are present in co-cultures. (D) ECAD cell–cell adhesion is present in all layers of the co-culture and colocalizes with β-catenin. (E) Cells in co-cultures with high (1.06 mM) CaCl₂ have morphologies (as assayed by F-actin using phalloidin) and cell–cell adhesion (as assayed by ECAD and PCAD) consistent with intact skin (Fig. S1). Phalloidin and ECAD: representative images from the bottom layer of co-culture. PCAD: representative image of bottom layer and top layer (inset). (C–E) Hoechst was used for nuclear stain. (F) Co-cultures maintained in low (0.06 mM) CaCl₂ do not have morphologies and cell–cell adhesion similar to intact skin (Fig. S1). (G) Co-cultured melanocyte dendrites (magenta, iRFPmem) interact with keratinocytes (green, EGFPmem) in the bottom and upper layers of the co-culture. Z, XY image from red line in maximum z projection. “s” corresponds to start of dendrite, and “f” corresponds to final point of the dendrite. Scale bars, (A) 15 µm; (B) 50 µm; (C) 25 µm; (D) 200 µm; (E) 25 µm; (F) 25 µm; and (G) 50 µm.