HPC viability measurement: Trypan blue versus acridine orange and propidium iodide

K. Mascotti, J. McCullough, Scott R. Burger

Research output: Contribution to journalArticlepeer-review

149 Scopus citations


BACKGROUND: A reliable, validated method for rapidly determining HPC viability is essential for clinical cell engineering. STUDY DESIGN AND METHODS: A fluorometric cell viability assay using acridine orange and propidium iodide (AO/PI) was compared to the current standard, trypan blue (TB) exclusion. Viable cells stained with AO/PI fluoresce green under darkfield fluorescence microscopy, while nonviable cells fluoresce orange. Mixtures of fresh and heat-killed bone marrow were prepared and used as viability standards for evaluation of both assays. The frequency of CFU-GM was determined for each specimen. RESULTS: Cell viability measured by AO/PI was extremely linear, with measured and predicted viability in agreement from 0 to 100 percent of the viable cells and a coefficient of regression (r2) of 0.9921. The predicted-viability regression line fell within the 95% CI for AO/PI-measured viability. The coefficient of regression for TB-measured viability was 0.9584, with the predicted-viability regression line almost entirely outside the 95% CI. TB overestimated the percentage of viable cells, particularly below the 50-percent level. CFU-GM frequency correlated better with cell viability measured by AO/PI (r2 = 0.979) than with that measured by TB (r2 = 0.930). CONCLUSIONS: The AO/PI viability assay is a rapid, highly linear, functionally correlated assay that is superior to conventional viability measurement by TB exclusion.

Original languageEnglish (US)
Pages (from-to)693-696
Number of pages4
Issue number6
StatePublished - 2000


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