Differentiation of EC cells in vitro by the fluorescent dye Hoechst 33342

The fluorescent dye Hoechst 33342 is able to differentiate F9 EC cells at low concentrations. This differentiation is accompanied by synthesis of large amounts of laminin, production of a well-developed cytoskeleton, disappearance of the SSEA-1 antigen, and synthesis of large amounts of fibronectin, all characteristics of the primitive endoderm. The dye immediately blocks the cells at the S/G2 phase of the cell cycle and produces a complete arrest in proliferation. This effect is not specific for the nullipotent F9 cell line, as multipotent EC cell lines like PCC3, P19, and PCC4 can also be easily differentiated into the same pathway by treatment with the Hoechst dye. In contrast, the dye has no remarkable effects on terminal differentiated, immortalized cells like NIH 3T3 or the parietal endoderm-like cell PYS-2.     

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.     

Nodular organization and differential intrametastatic distribution of the fluorescent dye Hoechst 33342 in B16 melanoma liver metastasis

The development of B16F10 liver metastases as related to its vascular organization was studied by morphological and functional methods, using the DNA-binding fluorochrome, Hoechst 33342. Experimental metastases were produced by intrasplenic injection of B16F10 melanoma cells, and the animals were sacrificed 3-7 days after tumor cell injection. The results show that early metastases are made up of avascular nodules of tumor cells, which subsequently developed lacunae which are not lined by endothelial cells and usually contain blood cells. A more developed metastasis seems to be made up of several nodules with or without lacunae. Between the nodules, vessels connected to blood circulation were usually observed. A functional study with a fluorescent dye showed that early metastases stained negatively, while more developed metastases showed a reticular fluorescent pattern, coincident with the intrametastatic vascular network and displaying a nodular image. In this case, the fluorescence displayed a gradient of intensity decreasing from the vessels towards the lacunae, where the tumor cells showed no fluorescence. In summary, our results suggest that B16 liver metastases have a nodular growth pattern, which is characterized by the formation, during their development, of lacunae not connected to the general circulation and that tumor cells have poor access to small molecules delivered from the blood.     

Fluorescence flow analysis of lymphocyte activation using Hoechst 33342 dye.

The in vitro response of murine lymphocytes to allogeneic and mitogenic stimulation has been studied by using the nontoxic fluorescent DNA probe, Hoechst 33342, and a fluorescence flow cytometer-cell sorter. Under appropriate conditions, two peaks of fluorescent intensity, not related to cellular DNA content, can be seen. As early as 12 hr after culture set up, lymphocyte activation can be identified. It appears that Hoechst-labeled lymphocytes of higher fluorescence intensity represent those cells activated by allogeneic stimulation whereas cells obtained from the lower intensity peak are nonresponding lymphocytes.     

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 analysis of the uptake of Hoechst 33342 dye by human lymphocytes

Human peripheral blood lymphocytes have been shown to resist staining with the DNA binding fluorochrome Hoechst 33342 by the cellular membrane. The rate of uptake of the dye is strongly temperature-dependent with minimal uptake rate below 16 degrees C. The activation energy of dye transport was found to be 135 kJ/mol above 20 degrees C and about 20 kJ/mol below 16 degrees C. Metabolic inhibitors accelerated, instead of inhibiting, the transport of the dye. Dead cells have been shown to stain promptly in contrast with the gradually staining viable cells. The uptake process in the vital staining conditions is suggested to involve a carrier mediated mechanism. Application of Hoechst 33342 as a fluorescent indicator of viability is proposed.     

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.     

Mechanism of interaction of DNA with fluorescent dye Hoechst 33258]

From the study of absorption and fluorescence spectra of the complex "Hoechst-33258"--DNA at different pH it is shown that AT--specific complex with DNA is formed by the neutral dye molecule, whereas the cationic state of the dye molecule forms the nonspecific complex. Possible formation of a specific complex in which the dye is bound to DNA in its major groove is discussed.     

Hoechst 33342: the dye that enabled differentiation of living X-and Y-chromosome bearing mammalian sperm

Hoechst 33342 is the fluorophore used routinely to measure DNA in X- and Y-chromosome-bearing mammalian sperm so they can be separated by flow sorting. A difference of <3% in DNA mass can be detected. This synthetic dye consists of two adjacent benzimidazole rings with N-methyl-piperazine and phenolic groups at the ends. The molecule permeates the cell membrane of living cells and binds selectively to A-T base pairs exposed in the minor-groove of double stranded DNA. Capability to distinguish and separate X- and Y-chromosome-bearing sperm has led to artificial insemination of somewhere around a million female mammals. Offspring with obvious abnormalities are no more frequent than after insemination of unsorted sperm into cows, horses, humans, pigs, sheep, rabbits, dolphins and other mammals. There is no apparent genotoxic effect from exposure of sperm to Hoechst 33342, although information on cellular toxicity or development of embryos resulting from Hoechst 33342-stained sperm is less reassuring. Little is known about the fate of sperm-delivered Hoechst dye in the female reproductive tract or on progeny of resultant offspring.     

Flow Cytometric Analysis of Marine Bacteria with Hoechst 33342

We investigated the accuracy and precision of flow cytometric (FCM) estimates of bacterial abundances using 4′, 6-diamidino-2-phenylindole (DAPI) and Hoechst 33342 (HO342, a bisbenzamide derivative) on paraformaldehyde-fixed seawater samples collected from two stations near Oahu, Hawaii. The accuracy of FCM estimates was assessed against direct counts by using epifluorescence microscopy. DAPI and HO342 differ in two aspects of their chemistry that make HO342 better suited for staining marine heterotrophic bacteria for FCM analysis. These differences are most important in studies of open-ocean ecosystems that require dual-beam FCM analysis to clearly separate heterotrophic bacterial populations from populations of photosynthetic Prochlorococcus spp. Bacterial populations were easier to distinguish from background fluorescence when stained with HO342 than when stained with DAPI, because HO342 has a higher relative fluorescence quantum yield. A substantially higher coefficient of variation of blue fluorescence, which was probably due to fluorescent complexes formed by DAPI with double-stranded RNA, was observed for DAPI-stained populations. FCM estimates averaged 2.0 and 12% higher than corresponding epifluorescence microscopy direct counts for HO342 and DAPI-stained samples, respectively. A paired-sample t test between FCM estimates and direct counts found no significant difference for HO342-stained samples but a significant difference for DAPI-stained samples. Coefficients of variation of replicate FCM abundance estimates ranged from 0.63 to 2.9% (average, 1.5%) for natural bacterial concentrations of 6 × 10⁵ to 15 × 10⁵ cells ml^-1.     

Comparison of Hoechst 33342 and propidium iodide as fluorescent markers for sperm fusion with hamster oocytes.

Hamster oocytes were loaded with the DNA dyes Hoechst 33342 or propidium iodide. Oocytes incubated in 10 mumol Hoechst 333421(-1) showed intracellular fluorescence within 10-20 s of exposure, as did hamster and guinea-pig spermatozoa. Impaled oocytes to which acrosome-intact hamster spermatozoa were bound before injection of Hoechst 33342 showed dye transfer to adhering spermatozoa within 2 min of injection. Oocytes loaded passively with Hoechst 33342 showed dye transfer to bound, acrosome-intact hamster spermatozoa within 10 min. On ultra-structural examination, no bound, acrosome-intact hamster spermatozoa (n = 311) were found to be fused. By contrast, oocytes incubated with 10 mumol propidium iodide l-1 showed no intracellular fluorescence after 2 h, although in approximately 50% of oocytes, fluorescence developed rapidly in the first polar body. Oocytes injected with propidium iodide showed intracellular fluorescence but no dye transfer to bound, acrosome-intact hamster spermatozoa. Oocytes impaled on pipettes containing propidium iodide showed no dye transfer to unlabelled oocytes with which they were brought into contact, whereas in similar experiments using Hoechst 33342 detectable dye transfer to an adjacent oocyte occurred within 10 min. Oocytes loaded with propidium iodide transferred propidium iodide to fusion-competent guinea-pig spermatozoa during in vitro fertilization. Normally, between 20 and 40 spermatozoa bound per oocyte, and the percentage of spermatozoa showing dye transfer varied between 0 and 41%. Dye transfer occurred within 5-45 min. Only those nuclei that showed propidium iodide transfer subsequently decondensed, suggesting that dye transfer is correlated with fusion. The presence of fused spermatozoa was confirmed by ultrastructural examination of oocytes. In separate experiments, hamster and guinea-pig spermatozoa showed detectable fluorescence from propidium iodide within 20 s of osmotic rupture or membrane stripping by detergent, suggesting the lag in dye transfer to sperm nuclei during fertilization reflects a delay in sperm-oocyte fusion following adhesion. This evidence suggests that Hoechst 33342 could be an unreliable marker for sperm-oocyte fusion in fertilization because of its capacity for passive movement from oocyte to spermatozoon. This problem can be overcome using oocytes injected with propidium iodide. With this technique, it was possible to show that fusion-competent guinea-pig spermatozoa that are held in pipettes will fuse with hamster oocytes when placed mechanically against the oocyte surface.     

Flow microfluorometric analysis of living spermatozoa stained with Hoechst 33342

Bovine spermatozoa were stained with Hoechst 33342. The fluorescence distribution of stained spermatozoa was complex. Non-motile spermatozoa displayed a higher fluorescence than did motile spermatozoa. The fluorescence profile of the motile spermatozoa was bimodal. Sort and reanalysis, and orientation experiments suggested that there are two distinct populations of motile spermatozoa.     

Measurement of cell proliferation in microculture using Hoechst 33342 for the rapid semiautomated microfluorimetric determination of chromatin DNA

We report the development and characterization of a semiautomated method for measurement of cell proliferation in microculture using Hoechst 33342, a non-toxic specific vital stain for DNA. In this assay, fluorescence resulting from interaction of cell chromatin DNA with Hoechst 33342 dye was measured by an instrument that automatically reads the fluorescence of each well of a 96-well microtiter plate within 1 min. Each cell line examined was shown to require different Hoechst 33342 concentrations and time of incubation with the dye to attain optimum fluorescence in the assay. In all cell lines, cell chromatin-enhanced Hoechst 33342 fluorescence was shown to be a linear function of the number of cells or cell nuclei per well when optimum assay conditions were employed. Because of this linear relation, equivalent cell doubling times were calculated from growth curves based on changes in cell counts or changes in Hoechst/DNA fluorescence and the fluorimetric assay was shown to be useful for the direct assay of the influence of growth factors on cell proliferation. The fluorimetric assay also provided a means for normalizing the incorporation of tritiated thymidine ( [3H] TdR) into DNA; normalized values of DPM per fluorescence unit closely paralleled values of percent 3H-labelled nuclei when DNA synthesis was studied as a function of the concentration of rat serum in the medium. In summary, the chromatin-enhanced Hoechst 33342 fluorimetric assay provides a rapid, simple, and reproducible means for estimating cell proliferation by direct measurement of changes in cell fluorescence or by measurement of changes in the normalized incorporation of thymidine into DNA.     

Microtubule changes during the development of generative cells in Hippeastrum vittatum pollen

Summary The three-dimensional organization of microtubules in generative cells during their development in pollen grains of Hippeastrum vittatum and the dynamic changes that occur were studied by collecting large quantities of fixed and isolated generative cells for immunofluorescence microscopy. The framework configuration and the arrangement pattern of the microtubule organization was investigated. The microtubule framework changed in shape from being spherical at an early stage to being long spindle-shaped at maturity: various transitional forms were observed: ellipsoidal, pear-shaped and short spindle-shaped. The microtubule arrangement making up this framework changed correspondingly from the original network, which was random in distribution, to axially oriented long bundles via an intermediate pattern composed of a mixture of networks with long bundles. However, cells with the same framework configuration might be heterogeneous in microtubule arrangements.     

Comparison of pollen transfer dynamics by multiple floral visitors: experiments with pollen and fluorescent dye

BACKGROUND AND AIMS: Most plant species are visited by a diversity of floral visitors. Pollen transfer of the four most common pollinating bee species and one nectar-robbing bee of the distylous plant Gelsemium sempervirens were compared. METHODS: Naturally occurring pollen loads carried by the common floral visitor species of G. sempervirens were compared. In addition, dyed pollen donor flowers and sequences of four emasculated recipient flowers in field cages were used to estimate pollen transfer, and the utility of fluorescent dye powder as an analogue for pollen transfer was determined. KEY RESULTS: Xylocopa virginica, Osmia lignaria and Habropoda laboriosa carried the most G. sempervirens pollen on their bodies, followed by Bombus bimaculatus and Apis mellifera. However, B. bimaculatus, O. lignaria and H. laboriosa transferred significantly more pollen than A. mellifera. Nectar-robbing X. virginica transferred the least pollen, even when visiting legitimately. Dye particles were strongly correlated with pollen grains on a stigma, and therefore provide a good analogue for pollen in this system. The ratio of pollen : dye across stigmas was not affected by bee species or interactions between bee species and floral morphology. However, dye transfer was more sensitive than pollen transfer to differences in floral morphology. CONCLUSIONS: The results from this study add to a growing body of literature highlighting that floral visitors vary in pollination effectiveness, and that visitors carrying the most pollen on their bodies may not always be the most efficient at depositing pollen on stigmas. Understanding the magnitude of variability in pollinator quality is one important factor for predicting how different pollinator taxa may influence the evolution of floral traits.     

Flow cytometric analysis of cell cycle-dependent changes in cell thiol level by combining a new laser dye with Hoechst 33342.

By halogenation of methylfluorescein-diacetate (MFDA) or eosin-diacetate, two new dyes for cellular thiol compatible with visible laser excitation have become available. These probes circumvent the use of an ultraviolet (UV)-excitation system as required by bimane-based dyes and allow combination with probes for other cellular parameters. The thiol dyes attain maximal staining after 10 min at 37 degrees C, and fluorescence is sensitive to pretreatment with diethylmaleate but not to buthionine sulfoximine. In a dual-laser system, analysis of the cellular thiol level as a function of cell cycle distribution can be achieved in viable cells by simultaneous staining with the bisbenzimidazole dye Hoechst 33342 and one of the halogenated dyes. Using this approach, we were able to show that cells in the G2 phase of the cell cycle were more sensitive to thiol depletion with diethylmaleate than were cells in the G1 compartment. The new thiol dyes allow a more flexible selection of wavelengths of excitation and emission for assessing changes in cellular thiol (glutathione and other thiol compounds) and allow this parameter to be examined as a function of cell cycle position.     

Fluorescence studies of Hoechst 33342 with supercoiled and relaxed plasmid pBR322 DNA

The fluorescence properties of Hoechst 33342 (HO 33342) were examined with plasmid pBR322 in the supercoiled (Form I) or relaxed covalently closed circular (Form Io) conformation in order to determine whether qualitative or quantitative differences in fluorescence properties might provide an assay for topological states of DNA. It was found that HO 33342 exhibited a 30% greater fluorescence intensity with Form I pBR322, independent of the dye or DNA concentration. As the dye to DNA ratio was increased, a red shift of approximately 8 nm was observed for HO 33342 complexed with Form I or Form Io. The red shift in fluorescence emission occurred at higher HO 33342 concentrations with Form I vs. Form Io DNA; however, when Form I and Form Io were mixed in various proportions, neither the fluorescent intensity differences nor the HO 33342 concentration at which the wavelength shift occurred could be used to quantitate the relative proportions of topological states present. These results suggest that although the fluorescence properties of HO 33342 complexed with Form I DNA are different than those of HO 33342 complexed with Form Io DNA, the fluorescence assay is not sufficiently sensitive to quantitatively discriminate among a mixture of DNA in various topological states.     

Chromatic shifts in the fluorescence emitted by murine thymocytes stained with Hoechst 33342.

BACKGROUND: Many methods in flow cytometry rely on staining DNA with a fluorescent dye to gauge DNA content. From the relative intensity of the fluorescence signature, one can then infer position in cell cycle, amount of DNA (i.e., for sperm selection), or, as in the case of flow karyotyping, to distinguish individual chromosomes. This work examines the staining of murine thymocytes with a common DNA dye, Hoechst 33342, to investigate nonlinearities in the florescence intensity as well as chromatic shifts. METHODS: Murine thymocytes were stained with Hoechst 33342 and measured in a flow cytometer at two fluorescence emission bands. In other measurements, cells were stained at different dye concentrations, and then centrifuged. The supernatant was then used for a second round of staining to test the amount of dye uptake. Finally, to test for resonant energy transfer, we measured fluorescence anisotropy at two different wavelengths. RESULTS: The fluorescence of cells stained with Hoechst 33342 is a nonlinear process that shows an overall decrease in intensity with increased dye uptake, and spectral shift to the red. Along with the spectral shift of the fluorescence to the longer wavelengths, we document decreases in the fluorescence anisotropy that may indicate resonant energy transfer. CONCLUSIONS: At low concentrations, Hoechst 33342 binds to the minor groove of DNA and shows an increase in fluorescence and a blue shift upon binding. At higher concentrations, at which the dye molecules can no longer bind without overlapping, the blue fluorescence decreases and the red fluorescence increases until there is approximately one dye molecule per DNA base pair. The ratio of the blue fluorescence to the red fluorescence is an accurate indicator of the cellular dye concentration.