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Merlin-deficient Schwann cells (MD-SC) and primary human vestibular schwannoma (VS) cells exhibit selective uptake of sodium-fluorescein (SF), allowing for fluorescent detection and improved visualization of tumor cells, when compared with Schwann cells (SC).SF is a fluorescent compound used for fluorescence-guided resection of gliomas. The utility of SF for VS surgery has not been assessed.Mouse MD-SCs and rat SCs were cultured on 96-well plates at different cell densities and treated with SF at several drug concentrations and durations. Relative fluorescence units (RFU) were measured using a fluorometer to determine optimal treatment parameters in vitro. Subsequently, a four-point Likert scale for fluorescence visualization of pelleted cells was created and validated. Blinded observers rated SF-treated primary human VS and SC cultures, which were developed from deidentified specimens obtained from live and cadaveric donors, respectively.In contrast to SCs that showed low levels of fluorescence, MD-SCs demonstrated dose-dependent increases in RFUs when treated with incremental dosages of SF as well as longer treatment and fluorescent excitation times. In addition, RFUs were higher at greater MD-SC densities. The Likert scale for fluorescence visualization was validated using nine blinded observers and there were excellent inter- and intrarater reliabilities (intraclass coefficients of 0.989 and >0.858, respectively). Using the Likert scale, human VS treated with SF received higher scores than human SCs (p < 0.001).Mouse MD-SC and human VS cells demonstrate preferential uptake of SF when compared with normal primary SCs. Observers detected differences in fluorescence using the validated Likert scale. Further investigations into the utility of SF-guidance in VS surgery are warranted.