The document discusses using different media to culture cells and measure yellow fluorescence over time. SC medium normally contains yellow fluorescent components. Cells were cultured in VD medium without riboflavin and their yellow fluorescence, peaking at 524nm, increased over time when excited at 430nm. The fluorescent material could pass through a dialysis membrane. A 3D fluorescent spectrum of the cell culture supernatant showed peaks from the excitation light. Using excitation between 500-525nm could avoid measuring yellow fluorescence from the supernatant.

1. SC medium contains yellow fluorescent components Alternative medium: VD(Vitamin Defined) medium without riboflavin Concentration of each component is the same as in the standard SC (synthetic complete) medium. [Filter settings] Excitation = 430 nm (for CFP) Emission = 530 nm (for YFP)

2. Yellow fluorescence increases even in VD medium Cells were arrested by use of alpha-factor. Fluorescent spectra of cell culture (VD-rivoflavin medium was used) were recorded periodically (excitation = 430nm). Yellow fluorescence (peak at 524nm) increased with time. This fluorescent material can go through dialysis membrane.

3. Three-dimensional fluorescent spectrum of supernatant from cell culture (Note) The sharp peaks are derived from the excitation light.

4. Three-dimensional fluorescent spectrum of supernatant from cell culture (Heat map display) If excitation light in the range (500 – 525 nm) is used it is possible to avoid the yellow fluorescence derived from the sup of cell culture.