Several concentrations of DMEM/F12 medium dissolving sodium were measured by freezing point osmotic measurement. Cells were seeded in 96-well plates at a density of 1 × 104 cells/well. Serum-free starvation was performed overnight, when cells reached 70%–80% confluence. Cells were then treated with serum-free medium at 312, 400, 450, 500, and 550 mOsm. After 24 h, the culture medium was removed and 100 μL basic culture medium containing 10% CCK-8 (Dojindo, Tokyo, Japan) was added; cells were incubated in the dark for up to 3 h and the absorbance (wavelength, in 450 nm) was measured by Infinite 200pro Microplate Reader. The absorbance value was recorded to determine the relative cell viability. After the background value had been subtracted, the resulting absorbance value was compared with that of the normal group. Several concentrations of CMC and α-MSH were pre-incubated for 30 min before hyperosmotic treatment. The same CCK-8 method was used to screen these concentrations.

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