Characterization and comparison of acridine orange/propidium iodide and acridine/DAPI viability detection methods for cell and gene therapy development

Abstract Cell viability is one of the critical quality attributes (CQAs) for analyzing and characterizing cellular therapeutic products. The characterization cell essential to ensure quality, safety efficacy during gene therapy development. In this work, we characterized compared two commonly used dual-staining methods, acridine orange/propidium iodide (AO/PI) orange/4′,6-diamidino-2-phenylindole (AO/DAPI) using Cellaca MX high-throughput counter. general, AO/DAPI method provided higher results than AO/PI, with measured difference between these methods ranging from 0% 24%. To identify sources difference, tested a list factors including dying process, staining time initial levels. First, observed larger naturally-dying Jurkat cells result heat-killed cells, suggesting that differences may depend on process. Second, found AO/PI more consistent count first 30 min time. Finally, showed might be low-viability, as healthy samples. our studies benefit selection are fit-for-purpose