Hoechst 33342 staining coupled with conventional histological technique

Hoechst 33342 was injected either intravenously or intraperitoneally into mice which were killed 1 or 24 hr or 7, 14 or 28 days later. Various organs were fixed and paraffin embedded. Visual inspection showed that independently of the route of dye administration or survival time, distinct fluorescence of nuclei was observed in organs other than cerebral cortex. Even formic acid decalcification of bone failed to abolish the fluorescence of osteocytes. In vivo staining with Hoechst 33342 is proposed as an alternative for staining after sectioning. Cells from spleens of Hoechst 33342-injected mice or stained in vitro were injected intramuscularly into mice. Hoechst 33342-stained splenocytes could be found in deparaffinized sections at the site of injection 24 hr later.     

Visualization of chromatin distribution in living PTO cells by Hoechst 33342 fluorescent staining

Chromatin distribution was visualized in living cells with the selective DNA fluorochrome Hoechst 33342. This dye was shown to be non-toxic on the rat kangaroo PTO cell line by measuring the labelled cell growth rate. The aim of this work was firstly to visualize chromatin distribution without fixation or dehydration and secondly to demonstrate that quantitative determination of DNA content was possible under these non-toxic labelling conditions. During interphase, condensed, decondensed and thin network chromatin configurations were visualized. In nucleolar regions the fluorochrome revealed well-defined chromocentres. During mitosis, fluorescent chromosome banding was observed in vital conditions and chromocentres on fixed chromosomes. Chromatin segregation was visualized after micronucleation, which induced chromosomal set distribution in individual micronuclei. By this means, we demonstrated that the chromocentres observed in interphase nuclei were part of nuclear organizer region (NOR)-bearing chromosomes. This vital staining of chromatin was shown to be compatible with the quantitative determination of DNA content, both in living PTO cells and in isolated nuclei.     

Effects of Hoechst 33342 on survival and growth of two tumor cell lines and on hematopoietically normal bone marrow cells

Our objective in this investigation was to determine whether Hoechst 33342, which is widely recognized as a DNA specific fluorochrome for living cells, is in fact nontoxic and kinetically nonperturbing at dye concentrations required to achieve acceptable DNA distributions. Three cell types were tested: HeLa S-3; SK-DHL2, a human lymphoma cell line; and hematopoietically normal human bone marrow cells. In the third system, only the cloning efficiencies were determined. Results differed considerably for the different cell types. While HeLa cells yielded excellent DNA distributions and were almost completely resistant to the cytotoxic and cytokinetic effects of the dye, SK-DHL2 cells were highly sensitive to the fluorochrome even at dye concentrations which produced very poor DNA distributions. Human bone marrow cells were intermediate in their stainability and toxicity response, and acceptable DNA distributions could be obtained at the nontoxic dye concentration of 2.5 muM. Clearly, different cell types differ considerably with respect to their cytotoxic and kinetic responses to Hoechst 33342. In some cases it may not be possible to ensure adequate staining of the cells for flow cytometry without significantly altering their viability and/or proliferative behavior.     

Effect of Hoechst 33342 staining on developmental competence of prepubertal goat oocytes

This study was undertaken to evaluate the effects of Hoechst staining on nuclear maturation and fertilisation when used at different stages of in vitro maturation (IVM) in prepubertal goat oocytes. Oocytes were matured in TCM1999 supplemented with 10% fetal bovine serum, 10 microg LH/ml, 10 microg FSH/ml and 1 microM 17beta-estradiol for 27 h. Frozen-thawed sperm cells were prepared by centrifugation in a discontinuous Percoll gradient and resuspended in DMH medium with 20% steer serum. Oocytes were fertilised in DMH medium with 7.75 mM calcium lactate. During IVM oocytes were exposed to 0.5 microg/ml of Hoechst 33342 staining and to ultraviolet light for a mean time of 3 s at 0 h, 8 h, 15 h, 20 h and 27 h. The percentage of metaphase II oocytes decreased significantly when oocytes were stained with Hoechst dye at 0 h, 8 h and 15 h of IVM. There was a decrease in total fertilisation rate and normal fertilisation rate of Hoechest-stained oocytes, independently of the time of Hoechst staining. Hoechst staining produces a significant reduction in oocyte viability when it is used in the early stages of in vitro maturation.     

Effect of drug efflux blockers on vital staining of cellular DNA with Hoechst 33342

The present study shows that staining of certain live cells, e.g., adriamycin-resistant P388 cells, by Hoechst 33342 is difficult because of the presence of a rapid efflux pump, which reduces intracellular dye concentration. Coincubation of these refractory cells in the presence of efflux blockers such as phenothiazines (trifluoperazine) or Ca++ channel blockers (verapamil) enhances dye retention and thus leads to generation of normal DNA distribution histograms. Laser flow cytometric data is confirmed by fluorometric assays, which show that P388/R cells retain one-third the amount of Hoechst 33342, and coincubation with efflux blockers increases Hoechst retention to values similar to those of drug-sensitive P388 cells. DNA histograms of mouse splenocytes incubated with Hoechst 33342 alone have a bimodal distribution possibly because of the presence of subpopulations that do not retain the fluorochrome owing to rapid efflux. Coincubation with an efflux blocker results in the generation of unimodal DNA histograms from these cells. These preliminary studies suggest that reduced retention of Hoechst 33342 in certain cell types (because of rapid efflux) can be blocked by efflux blockers, thus leading to generation of typical DNA distribution histograms.     

Equilibrium binding of Hoechst 33258 and Hoechst 33342 fluorochromes with rat colorectal cells

We examined the biophysical characteristics of the interaction of Hoechst 33258 and 33342 dyes with normal rat colorectal cells as functions of fixation and solution composition. Classical dye-binding techniques were used to investigate the stoichiometry and binding constants with whole cells, and quantitative fluorescence image analysis was used to specifically study nuclear dye binding in intact cells. In aqueous solution, H-33258 dye bound cooperatively with intact cells, with a binding constant of between 3-4 x 10(5). In ethanolic solution, binding appeared less cooperative, although Scatchard analysis could not be used. The binding constant was slightly lower (2 x 10(5)), but the total number of cell binding sites was decreased by a factor of 5, reflecting a great decrease in cytoplasmic sites. QFIA studies identified conditions optimal for DNA quantitation under which the fluorescence signal was independent of dye or cell concentration. The proportionality between absolute nuclear fluorescence intensity and DNA content was established, and the upper limit of DNA content of normal colorectal cells was also determined.     

Insertion of the bacterial gene for dihydrofolate reductase into colony-forming cells of mouse bone marrow

Introduction of the plasmid containing the methotrexate-resistant (Mtx-r) bacterial gene of dihydrofolate reductase (DHFR) under the control of the early promoter of SV 40 into the donor bone cells of the mouse with subsequent transplantation of the cells into lethally irradiated mice results in the increase in the life span of mice under conditions of methotrexate selection. It is due to the stable transformation of the bone marrow colony-forming cells with the plasmic DNA and the synthesis of the bacterial Mtx-r DHFR in the spleen and bone marrow of the recipient mouse.     

Clastogenic effects of acrylamide in mouse bone marrow cells

Acrylamide, known to induce dominant-lethal mutations (Shelby et al., 1986; Smith et al., 1986) and heritable translocations (Shelby et al., 1987) in rodent germ cells, was hitherto a questionable clastogen in rodent bone marrow (Shiraishi, 1978). Therefore, it was tested for chromosomal aberrations in mouse bone marrow cells, spermatogonia and by the micronucleus test. The intraperitoneally injected doses ranged from 50 to 150 mg/kg. In the chromosomal bone marrow test and the micronucleus assay positive results were obtained with acrylamide, and in the latter test the effect increased linearly with dose. Chromosomal aberrations were not induced in differentiating spermatogonia by the acute acrylamide treatment. Cisplatin was used as a positive control and gave the expected positive response in all 3 tests. The present results demonstrate that acrylamide is no exception among clastogens. It breaks chromosomes not only in mammalian germ cells but also in somatic cells.     

Cytogenetic effects of dacarbazine on mouse bone marrow cells in vivo

The mutagenicity of dacarbazine was assayed in an in vivo test utilizing mouse bone marrow cells. The dose rates used in the experiments were computed according to the standard surface area of the mouse and were proportional to the human dose rate. These were 0.27, 0.44 and 0.60 mg/30 g body weight, each given twice daily at an interval of not less than 6 h. The duration of drug treatment was 24, 48 and 72 h. This alkylating agent proved to be mitodepressive and produced a 3-fold reduction in the mitotic index. The drug also induced chromosome anomalies mainly in the form of chromatid gaps and breaks. These anomalies were proportional to dose rate and duration of drug treatment.     

Proliferative and differentiation potentials of skeletogenic bone marrow colony-forming cells

The clonal nature of bone marrow fibroblast colonies derived from clonogenic bone marrow osteogenic cells (CFUf) was proved by the chromosome analysis. During subsequent passages of multi-colony derived bone marrow fibroblast strains there occurs a pronounced increase in the cell number and in the number of osteogenic units (tested by transplantation in diffusion chambers). Single colony-derived strains are capable of forming bone and cartilage simultaneously. It follows that CFUf or part of them are clonogenic cells with high proliferative potentials and are common precursors for bone and cartilage tissue. Thus, CFUf may be regarded as osteogenic stem cells.     

Cell cycle dependence of Hoechst 33342 dye cytotoxicity on sorted living cells

The effect of staining cellular DNA with the bisbenzimidazole dye Hoechst 33342 on the colony forming efficiency of Chinese Hamster Ovary Cells in different cell cycle phases has been studied. Exposures of 90 and 120 min to 5 microM Hoechst 33342 provided a considerable loss of clonogenicity depending on the cycle phase at staining procedure. The G2+M cells reveal to be the most sensitive fraction followed by the G1 cells. The highest resistance was found on S-phase cells with a colony forming efficiency exceeding that of the G2+M fraction by a factor of two.     

Flow cytometric characterization of viable meiotic and postmeiotic cells by Hoechst 33342 in mouse spermatogenesis.

BACKGROUND: Spermatogenesis in adult is a complex stepwise process leading to terminally differentiated spermatozoa. The cellular heterogeneity of testis renders complex the studies on molecular aspects of this differentiation process. Analysis of the regulation of adult spermatogenesis would undoubtedly benefit from the development of techniques to characterize each germinal differentiation step. METHODS: Hoechst 33342 staining of mouse testicular cells allows characterization of an enriched population in germinal stem cell and spermatogonia, called side population. In this study, we examined the definition of the various germinal populations stained by Hoechst 33342, notably meiotic and postmeiotic cells. RESULTS: Preleptotene spermatocytes, spermatocyte I, spermatocyte II, and round and elongated spermatids were discriminated by Hoechst 33342 staining. In addition, we associated differentiation of spermatocyte I through leptotene to diplotene with changes in Hoechst 33342 red fluorescence pattern. CONCLUSIONS: Hoechst 33342 staining of viable germinal cells constitutes a valuable tool to study normal and impaired mouse adult spermatogenesis or to isolate viable cells from various differentiation stages for studies of molecular mechanisms regulating spermatogenesis.     

Cell cycle dependence of Hoechst 33342 dye cytotoxicity on sorted living cells

Summary The effect of staining cellular DNA with the bisbenzimidazole dye Hoechst 33342 on the colony forming efficiency of Chinese Hamster Ovary Cells in different cell cycle phases has been studied. Exposures of 90 and 120 min to 5 M Hoechst 33342 provided a considerable loss of clonogenicity depending on the cycle phase at staining procedure. The G2+M cells reveal to be the most sensitive fraction followed by the G1 cells. The highest resistance was found on S-phase cells with a colony forming efficiency exceeding that of the G2+M fraction by a factor of two.     

Clastogenic effects of cesium chloride on mouse bone marrow cells in vivo

Clastogenic effects of cesium chloride (CsCl) on mouse bone marrow cells in vivo following oral administration were studied after 24 h. The incidence of chromosome aberrations increased linearly with increasing concentrations of the chemical from 1/20th to 1/5th of the LD50. The frequency of cell division was also enhanced by the lower doses but higher doses were mitostatic. This report is the first on the clastogenicity of cesium on animals.     

Effects of Hoechst 33342 staining and ultraviolet irradiation on the developmental competence of in vitro-matured porcine oocytes

Hoechst 33342 (H342) in combination with ultraviolet (UV) irradiation is frequently used to assist the enucleation of porcine oocytes in somatic cell nuclear transfer programs. This work evaluated the effects of H342 (5 μg/mL for 12 min) staining and/or exposure to UV irradiation on fertilisability and developmental capacity of porcine oocytes matured in vitro. In Experiment 1, a total of 1388 mature oocytes were distributed in the following groups: Group 1: oocytes without treatment (Control), Group 2: oocytes stained with H342, Group 3: oocytes stained with H342 and UV irradiated for 30 sec, and Group 4: oocytes UV irradiated for 30 sec. Oocytes from each group were exposed to thawed spermatozoa and cultured for 18 h to assess fertilization parameters or for 7 d to evaluate embryo development. Sperm penetration (P < 0.001) and monospermy (P < 0.04) were lower in oocytes exposed to H342/UV (80.7 ± 4.5% and 30.7 ± 5.4%, respectively) than in oocytes from the control group (94.9 ± 4.3 and 50.0 ± 4.9, respectively). The oocytes exposed to H342/UV showed lower (P < 0.001) cleavage (49.8 ± 2.9%) and blastocyst (7.7 ± 2.9%) rates than oocytes from the other groups (range: 73.8 ± 2.9% to 77.7 ± 2.9% and 22.3 ± 2.9% to 30.9 ± 3.0%, respectively). Experiment 2 was designed to evaluate the effect of shorter UV irradiation (5 sec). A total of 1835 mature oocytes were separated into the same groups as those of Experiment 1. The fertilization parameters and the cleavage rates were not influenced by the different treatments. However, the oocytes exposed to H342 and UV irradiation for 5 sec showed a lower (P < 0.02) rate of blastocyst formation (15.2 ± 4.5%) than the oocytes from other groups (range: 26.1 ± 4.5% to 30.7 ± 4.5%). In conclusion, our results demonstrate that the combination of H342 staining with UV irradiation has a clear deleterious effect on the developmental ability of oocytes, with the effects being more intense with increased exposure to UV irradiation.     

Effects of Hoechst 33342 staining and ultraviolet irradiation on the developmental competence of in vitro-matured porcine oocytes

Hoechst 33342 (H342) in combination with ultraviolet (UV) irradiation is frequently used to assist the enucleation of porcine oocytes in somatic cell nuclear transfer programs. This work evaluated the effects of H342 (5 μg/mL for 12 min) staining and/or exposure to UV irradiation on fertilisability and developmental capacity of porcine oocytes matured in vitro. In Experiment 1, a total of 1388 mature oocytes were distributed in the following groups: Group 1: oocytes without treatment (Control), Group 2: oocytes stained with H342, Group 3: oocytes stained with H342 and UV irradiated for 30 sec, and Group 4: oocytes UV irradiated for 30 sec. Oocytes from each group were exposed to thawed spermatozoa and cultured for 18 h to assess fertilization parameters or for 7 d to evaluate embryo development. Sperm penetration (P < 0.001) and monospermy (P < 0.04) were lower in oocytes exposed to H342/UV (80.7 ± 4.5% and 30.7 ± 5.4%, respectively) than in oocytes from the control group (94.9 ± 4.3 and 50.0 ± 4.9, respectively). The oocytes exposed to H342/UV showed lower (P < 0.001) cleavage (49.8 ± 2.9%) and blastocyst (7.7 ± 2.9%) rates than oocytes from the other groups (range: 73.8 ± 2.9% to 77.7 ± 2.9% and 22.3 ± 2.9% to 30.9 ± 3.0%, respectively). Experiment 2 was designed to evaluate the effect of shorter UV irradiation (5 sec). A total of 1835 mature oocytes were separated into the same groups as those of Experiment 1. The fertilization parameters and the cleavage rates were not influenced by the different treatments. However, the oocytes exposed to H342 and UV irradiation for 5 sec showed a lower (P < 0.02) rate of blastocyst formation (15.2 ± 4.5%) than the oocytes from other groups (range: 26.1 ± 4.5% to 30.7 ± 4.5%). In conclusion, our results demonstrate that the combination of H342 staining with UV irradiation has a clear deleterious effect on the developmental ability of oocytes, with the effects being more intense with increased exposure to UV irradiation.     

Cytogenetic effects of ribavirin on mouse bone marrow

The micronucleus test and mitotic chromosome analysis were used to study the in vivo mutagenic activity of ribavirin on bone marrow cells of Swiss albino mice. To determine the incidence of micronuclei, mice were injected i.p. twice, at an interval of 24 h. with the drug at doses of 20, 100 and 200 mg/kg. Animals were killed 6 h after the second dose and bone marrow was examined for the presence of micronuclei in developing erythrocytes. Ribavirin significantly (P less than 0.05) induced micronuclei in polychromatic erythrocytes at all doses. A study was conducted to investigate the cytogenetic effect of the drug on mitotic chromosomes. Ribavirin at 200 mg/kg/day was administered to mice for 3 and 5 days. Repeated treatment with the high dose of ribavirin produced a highly significant (P less than 0.02) increase in abnormal metaphase spreads. The results indicate that ribavirin is mutagenic to bone marrow cells of mice as evaluated by the micronucleus test and by chromosome analysis.