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

Abstract

BackgroundSimultaneous 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.FindingsIn 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.ConclusionsSYTO 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.

Novel Application of Stem Cell-Derived Neurons to Evaluate the Time- and Dose-Dependent Progression of Excitotoxic Injury

“…The pairing of fluorescent membrane-permeant and impermeant nuclear dyes was used to evaluate the mode of excitogenic cell death (Figure 7A) [20]. Tonic addition of 3–200 μM glutamate resulted in pyknotic, PI-negative nuclei at 2 h, indicative of apoptosis (Figure 7B).…”

Head-to-Head Comparison of the Penetration Efficiency of Lipid-Based Nanoparticles into Tumor Spheroids

“…Fully formed spheroids were rinsed three times with imaging medium and were treated with nanoparticles in a fresh imaging medium for 5 h. After 3 h of incubation, the spheroids were nuclear-labeled by supplementing the wells with a cell-permeable nucleic acid stain (SYTO13, Invitrogen; 488/509 nm). Although the nuclear stain is a high-performance dye, it causes phototoxicity within 3 h, 59 which restricts live-cell imaging. Spheroids were washed three times in PBS (Gibco) and were then immersed in a fresh imaging medium.…”

A Fluorescent Real-Time Plaque Assay Enables Single-Cell Analysis of Virus-Induced Cytopathic Effect by Live-Cell Imaging

“…DNA fluorescent dyes have differential cell permeability properties. Hoechst 33342 (referred hereafter as Hoechst) is typically used in fluorescence microscopy and flow cytometry to stain cell nuclei and chromatin condensation, an early marker of cell death since it is able to cross cell membranes of both living and dying cells [15,16]. In addition, cell-impermeant dyes such as SYTOX Green and propidium iodide (PI), are used to stain the nuclei of dead cells with terminal membrane permeabilization, which correlates with the final stages of a viral cytopathic effect [10,17].…”