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.


RESULTS
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.
United States Army Medical Research Institute of Chemical Defense, 3100 Ricketts Point Rd, Aberdeen Proving Ground, MD 21010, USA.
Kyle S
KS
Hubbard
Ian M
IM
Gut
Stephen M
SM
Scheeler
Megan E
ME
Lyman
Patrick M
PM
McNutt
D000818:Animals; D001562:Benzimidazoles / Q000032:analysis / Q000494:pharmacology*; D016923:Cell Death / Q000187:drug effects; D002454:Cell Differentiation; D002463:Cell Membrane Permeability; D002467:Cell Nucleus / Q000187:drug effects / Q000648:ultrastructure; D053145:Cell Nucleus Size / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D053595:Embryonic Stem Cells / Q000166:cytology; D005456:Fluorescent Dyes / Q000032:analysis / Q000494:pharmacology*; D051379:Mice; D008856:Microscopy, Fluorescence; D057054:Molecular Imaging / Q000379:methods*; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000648:ultrastructure; D009930:Organic Chemicals / Q000032:analysis / Q000494:pharmacology; D059010:Single-Cell Analysis / Q000379:methods*; D019311:Staurosporine / Q000494:pharmacology; D013997:Time Factors; D059008:Time-Lapse Imaging
D001562:Benzimidazoles; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C461159:SYTO 13; D019311:Staurosporine; C017807:bisbenzimide ethoxide trihydrochloride