Compatibility of SYTO 13 and Hoechst 33342 for longitudinal imaging of neuron viability and cell death

BACKGROUND: Simultaneous use of cell-permeant and impermeant fluorescent nuclear dyes is a common method to study cell viability and cell death progression. Although these assays are usually conducted as end-point studies, time-lapse imaging offers a powerful technique to distinguish temporal changes in cell viability at single-cell resolution. SYTO 13 and Hoechst 33342 are two commonly used cell-permeant nuclear dyes; however their suitability for live imaging has not been well characterized. We compare end-point assays with time-lapse imaging studies over a 6 h period to evaluate the compatibility of these two dyes with longitudinal imaging, using both control neurons and an apoptotic neuron model. FINDINGS: In longitudinal assays of untreated neurons, SYTO 13 addition caused acute necrosis within 3 h, whereas neurons imaged with Hoechst remained viable for at least 6 h. In a staurosporine-induced apoptotic model of neurotoxicity, determinations of the mode of cell death and measurements of nuclear size were identical between longitudinal studies using Hoechst and end-point assays. Alternatively, longitudinal studies using 500 nM or 5 nM SYTO 13 were not consistent with end-point assays. CONCLUSIONS: SYTO 13 is acutely neurotoxic and when used in longitudinal studies, masked end-stage morphologic evidence of apoptotic cell death. In contrast, a single application of Hoechst evoked no evidence of toxicity over a 6 h period, and was consistent with end-point characterizations of cell viability and nuclear morphology. For longitudinal characterization of acute cell death, Hoechst is a superior option.