A1345

The pH values of bovine, porcine, and chicken MSC spent differentiation media were measured on days 0–2 and 2–4 in the presence of an antioxidant supplement (Sigma-Aldrich, St. Louis, MO, USA) or quercetin using a pH meter (Mettler Toledo, Columbus, OH, USA). The antioxidant supplement is a commercialized product used in different types of cell cultures as a cocktail containing various antioxidants (Luo et al., 2014 (link); Tai et al., 2012 (link)). Sigma-Aldrich's proprietary antioxidant supplement (A1345) can be used in the diversity of cell culture including primary cell culture. Therefore, the antioxidant supplement (Sigma-Aldrich, A1345) is already in the market and is considered suitable for use as a positive control to confirm the antioxidant effect that is used in this study.

Primary cells were isolated from whole epidermises of 2 days old WT, CRTC1 KO, CRTC2 KO and CRTC3 KO pups and cultured in RPMI media containing TPA (P1585, Sigma) and cholera toxin (C8052, Sigma), following previously described detailed protocol (Godwin et al., 2014 (link)). For skin melanoblast quantification, WT and CRTC3 KO mice were crossed with mice carrying iDCT:GFP transgene system. Pregnant females were given 1mg/ml doxycycline in water and epidermal cell suspensions were isolated from 2 days old pups (WT N = 5, KO N = 4). GFP positive cells were counted through fluorescence-activated cell sorting (Becton Dickinson Influx cytometer). To account for differences in total amounts of cells isolated from each animal, quantification was expressed as percentage of GFP positive cells per epidermis. In experiments where XB2 feeder keratinocytes were used, cells were obtained from Wellcome Trust Functional Genomics Cell Bank and prepared using mitomycin c treatment (M4287, Sigma), as previously described (Godwin et al., 2014 (link)). In some experiments, catalase (LS001896, Worthington Biochemical) and an antioxidant supplement (A1345, Sigma) were added to the primary cultures in the attempt to improve survival and differentiation of CRTC3 KO melanoblasts.

The media of bovine, porcine, and chicken MSCs differentiated for 4 days in the presence of antioxidant supplement (Sigma-Aldrich, A1345) or quercetin was removed and treated with 10 μM 2',7'-dichlorofluorescein (Sigma-Aldrich, St. Louis, MO, USA) for 2 h at 37 °C. After washing twice with PBS, fluorescence was measured (excitation 498 nm and emission 522 nm) using a microplate reader (BioTek, SynergyTM H1 Hybrid Multi-Mode Microplate Reader, Winooski, VT, USA).