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

We developed a rapid technique for preservation of Hoechst 33342/propidium iodide-stained cells, using ethanol as a fixative. Combined staining with these dyes makes possible analysis of cell-cycle phase-specific cell death. The technique relies on exclusion of propidium iodide from the viable cells, whereas Hoechst stains all of the cells. The bivariate histograms resulting from the flow cytometric analysis contain the equivalent of two single-parameter DNA histograms, one of the living and the other of the dead cell population. Preservation of staining involved addition of 25% ethanol in PBS after propidium iodide staining and before Hoechst staining. The separation between the living and the dead cell populations was maintained for over 3 days at 4 degrees C. This technique will be valuable for quantitative evaluation of the cell-cycle phase-specific effects of cytostatic or cytotoxic agents, particularly in situations where a lag period between staining and analysis is unavoidable.

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An in vitro study of Hoechst 33342 redistribution and its effects on cell viability

Human & Experimental Toxicology ◽

10.1191/0960327105ht570oa ◽

2005 ◽

Vol 24 (11) ◽

pp. 573-580 ◽

Cited By ~ 13

Author(s):

N Mohorko ◽

N Kregar-Velikonja ◽

G Repovs ◽

M Gorensek ◽

M Bresjanac

Keyword(s):

Cell Death ◽

Cell Transplantation ◽

Fluorescence Intensity ◽

Time Course ◽

In Vitro Study ◽

Host Cells ◽

Hoechst 33342 ◽

Overnight Incubation ◽

Donor Cells

Although Hoechst 33342 (H342) is frequently used to label donor cells in cell transplantation research, it has been noted that it might secondarily label the host cells. Furthermore, its potential toxicity leading to cell death has been described. We studied the time course of H342 redistribution from the primary labeled rat bone marrow stromal cells (rBMSC) into the non-labeled rBMSC population over 7 days in culture; we evaluated the nuclear H342 fluorescence intensity as a possible criterion for distinguishing the primary from the secondary labeled cells, and determined the viability of rBMSC after an overnight incubation in 1 mg/mL of H342. H342 labeled / 50% of the initially non-labeled cells within the first 6 hours and almost 90% within a week.Nuclear fluorescence intensity was a reliable criterion for distinguishing primary and secondary labeled cells within the first 24 hours, but less so at later time points. The percentage of either apoptotic or necrotic cells did not rise acutely after the overnight incubation in 1 mg/mL of H342. Although a 12-hour incubation of rBMSC in 1 mg/mL of H342 did not cause acute cell death, H342 rapidly and extensively redistributed into non-labeled cells, which makes H342 a relatively unsuitable marker for cell transplantation research.

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Effects of Fungicide Euparen Multi (Tolylfluanid) on Development of Preimplantation Embryos in Mouse

Acta Veterinaria Brno ◽

10.2754/avb200776020209 ◽

2007 ◽

Vol 76 (2) ◽

pp. 209-214 ◽

Cited By ~ 3

Author(s):

M. Domaracký ◽

P. Rehák ◽

J. Legáth ◽

J. Koppel

Keyword(s):

Cell Death ◽

Preimplantation Embryo ◽

Preimplantation Embryos ◽

Hoechst 33342 ◽

Preimplantation Embryo Development ◽

Cell Numbers ◽

Calcein Am ◽

Dose Dependent ◽

Triple Staining ◽

Different Doses

The effect of the fungicide Euparen Multi (containing 50% tolylfluanid) on the development of mouse preimplantation embryos was evaluated. Euparen Multi was daily administered per os to female mice (ICR strain) at four different doses of 118, 294, 588 and 1177 mg/kg b.m., beginning on day 1 of pregnancy. Embryos obtained on day 4 of pregnancy were stained by morphological triple staining (Hoechst 33342, propidium iodide, Calcein AM), and the number of nuclei, blastocyst formation, distribution of embryos according to the nucleus number and cell death incidence were determined. Embryos in the experimental groups (except for the lowest dose 118 mg/kg b.m.) showed a highly significant dose-dependent reduction in total cell numbers corresponding to the lower proportion of blastocysts. The occurrence of cell death was significantly increased in all experimental groups, indicating that Euparen Multi is able to cause cell death at relatively low doses. Our data demonstrate that Euparen Multi could induce significant alterations in the preimplantation embryo development.

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