Melanocytes co-cultured with keratinocyte have local fluctuations in cytosolic Ca²⁺ in the absence of exogenous growth factors. (A) The majority of co-cultured melanocytes, in the absence of exogenous growth factors, have at least one Ca²⁺ transient when co-cultured with keratinocytes (n = 22 co-cultures; color indicates skin donor, n = 6). See also Video 1. Of the melanocytes with transients, the majority had local Ca²⁺ transients, while few had global whole cell Ca²⁺ transients. (B) Local Ca²⁺ transients occur at different regions and different times within GCaMP6f expressing melanocytes in co-culture with keratinocytes. (C) The majority of local Ca²⁺ transients are in melanocyte dendrites (n = 3 co-cultures from three different donors randomly chosen from A). (D) Representative cell body Ca²⁺ transient that is localized to a small region of the cell body with corresponding ΔF/F0 plot from ROI at the center of the of the transient. (E) Representative whole cell Ca²⁺ transient with corresponding ΔF/F0 plot from designated regions: CB, cell body; DX, primary dendrites where X indicates dendrite number; DX-CB, dendrite–cell body junction; DX-by, secondary dendrites; y indicates secondary branch number. (F) Melanocytes in co-cultures with 10× keratinocytes (10xKer, the normal ratio of keratinocyte to melanocytes in co-cultures) have significantly more Ca²⁺ transients than those co-cultured with HEK293Ts (10× 293T), alone (Mel, normal melanocyte seeding density but no added keratinocytes), or with 10× melanocytes (10xMel, GCaMP6f melanocytes culture with an additional 10× melanocytes not expressing GCaMP6f); n = 3 co-cultures per condition; colors indicate different skin donors. One-way ANOVA F(3,8) = 29.05, P = 0.00012; post hoc Tukey means comparison to other culture conditions, **, P = 0.005. See also Fig. S2. (G) Distribution of the number of transients per cell for co-cultured melanocytes (data from A and one additional donor; n = 1,793 melanocytes from 27 co-cultures from seven different skin donors) and mono-cultured melanocytes (inset, n = 563 melanocytes from five co-cultures of different skin donors). (H) Distribution of the spatial spread of the Ca²⁺ signal (full-width half maximum Gaussian fit) at peak ΔF/F0 intensity for transients within the dendrites of co-cultured melanocytes (n = 53 dendrites from 11 co-cultures from three different skin donors). For all Ca²⁺ imaging, growth media were removed, and cultures were washed three times then imaged in a modified DPBS solution with no growth factors or serum. Scale bars, (B) 25 µm, top, 2 µm, bottom, 5 µm; (D) 75 µm; (E) 100 µm.