Transplantation of human cells after isolation and culture has become an important alternative for treatment of acute or chronic skin wounds. To increase the efficacy and reduce cost for transplantation of skin cells, more efficient and accurate techniques for evaluation of cell proliferation are needed. Hemocytometer counts provide a valid assessment of cell proliferation and viability, but they are very labor intensive and require removal of the cells from their substrate. In this study, hemocytometer counts were compared with a fluorometric assay (n = 21 per condition) that uses the commercially available reagent alamarBlue, which is reduced to a fluorescent substrate by cellular dehydrogenases. Human epidermal keratinocytes were inoculated at 200, 600, 2000, and 6000 cells/cm2 incubated for 6 days in modified MCDB 153 medium. Alamar Blue was incubated with cells for 2 h at 37 degrees C, and fluorescence was measured with a microplate reader at 590 nm. Hemocytometer counts (x10(-4)) from the respective cell inoculation densities were 0.30 +/- 0.04, 1.07 +/- 0.10, 6.37 +/- 0.62, and 16.99 +/- 0.96. Fluorescence values (x10(-3)) for the respective inoculation densities were 0.14 +/- 0.01, 0.34 +/- 0.02, 1.20 +/- 0.09, and 1.79 +/- 0.12. Regression analysis showed a statistical significant (p < 0.0001) correlation (r2 = 0.87) between cell counts and optical density from the alamarBlue assay. These data demonstrate that alamarBlue provides a valid substitute for cell counts to assess cell proliferation before clinical transplantation of engineered skin. AlamarBlue also allows repeated, nondamaging assessment of living cells over time. These advantages are expected to increase the validity and reliability of quality assurance standards for transplanted skin cells, and to increase the efficacy of healing of cutaneous wounds.