FMN Measurement Calibration

Riboflavin 5′-monophosphate sodium salt hydrate (Sigma-Aldrick, United States) was dissolved in 0.9% sodium chloride to make a series of FMN solutions with concentrations ranging from 12.5 to 625.0 ng/mL. Two hundred microliters of each solution in triplicates were loaded into a black nonbinding 96-well microplate (Greiner Bio-one, Austria) at room temperature under dimmed light. The microplate was analyzed by fluorescence spectroscopy (Infinite 200 PRO, Switzerland). A monochrome light with excitation wavelength of 450 nm was used and the fluorescence with wavelength of 525 nm emitted by the FMN was recorded with 100% gain. The fluorescence readings in artificial unit (AU) were displayed in an Excel spreadsheet produced by the spectrometer. This experiment was performed 3 times. The average fluorescence readings of the FMN solutions were plotted against their known concentrations to generate a standard curve. The same steps were repeated for the FMN solutions that were stored at 4°C for 1 day and 7 days and at –80°C for 28 days, to assess whether cold storage alters FMN concnentration.