Ab277270

Quantitative assessments for melanosome transfer in MC-KC co-culture. Cells in co-culture were treated with the indicated compounds for 48 h. To quantitatively assay melanosome transfer. The co-cultured cells were harvested and washed with cold PBS, fixed in 4% paraformaldehyde for 10 min, washed with PBS containing 0.1% Triton-X100 for 5 min. MCs were immunostained with anti-TRP1 antibody (ab178676, Abcam, Cambridge, UK) and goat anti-mouse IgG H&L/PE secondary antibody (bs-0296G-PE, Bioss, Beijing, China) and KCs were incubated with anti-pan cytokeratin antibody conjugated with alexa fluor 488 (ab277270, Abcam, Cambridge, UK). Stained cells were analysed by flow cytometry. A total of 10 000 events were collected on the CytoFLEX Flow Cytometer (Beckman coulter, Brea, CA, USA). The ratio of percentage of cells positive for both cytokeratin and tyrosinase to that of cells positive for cytokeratin only represents melanosome transfer efficacy [30 (link),34 (link),35 (link)].

The co-cultured cells were washed with ice-cold PBS, fixed in 4% paraformaldehyde for 10 min, and washed with PBS containing 0.25% Triton-X100 for 10 min to permeabilize the membrane. Add blocking buffer (Beyotime, Shanghai, China) for 10 min at 37 ℃. Then MCs were immunostained with anti-TRP1 antibody (ab178676, Abcam, Cambridge, UK) and goat anti-mouse IgG H&L/PE secondary antibody (bs-0296G-PE, Bioss, Beijing, China) and KCs were incubated with anti-pan cytokeratin antibody conjugated with alexa fluor 488 (ab277270, Abcam, Cambridge, UK). Nucleus were stained with DAPI. Images were obtained with an Inverted fluorescence microscope (Mshot, Guangzhou, China) and dealt with Image pro plus [36 (link)].