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Updated: Aug 10, 2026
Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture
Published on: May 10, 2017
HPC viability measurement: trypan blue versus acridine orange and propidium iodide
K Mascotti1, J McCullough, S R Burger
1Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
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 (r(2)) 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 (r(2) = 0.979) than with that measured by TB (r(2) = 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.
