HPC viability measurement: trypan blue versus acridine orange and propidium iodide.
BACKGROUND
A reliable, validated method for rapidly determining HPC viability is essential for clinical cell engineering.


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.
Mascotti
K
K
Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
McCullough
J
J
Burger
S R
SR
D000165:Acridine Orange / Q000032:analysis* / Q000378:metabolism; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism; D002463:Cell Membrane Permeability; D002470:Cell Survival; D005069:Evaluation Studies as Topic; D005456:Fluorescent Dyes / Q000032:analysis* / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D011419:Propidium / Q000032:analysis* / Q000378:metabolism; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods*; D014343:Trypan Blue / Q000032:analysis* / Q000378:metabolism
D005456:Fluorescent Dyes; D011419:Propidium; D000165:Acridine Orange; D014343:Trypan Blue