NaCl-aided Hoechst 33258 staining method for DNA quantification and its application

Summary We investigated the effect of salt on the fluorescence staining procedure for quantification of the amount of DNA in cell nuclei in situ. For this, NaCl was added at various concentrations to the Hoechst 33258 fluorochrome (Hoe) medium for staining DNA. The fluorescence intensity of free DNA-Hoe solution was not changed by the addition of NaCl, but that of the nuclei-Hoe complex in situ increased 4-fold on increasing the NaCl concentration up to 1 M. SDS polyacrylamide gel electrophoresis showed that histones H1, H2A, and H2B dissociated from cell nuclei in the presence of 1 M NaCl, resulting in increasing accessibility of DNA to the fluorochrome.The applicability of the NaCl-aided fluorescence staining method was evaluated by measuring the ploidy classes of various cells. The amount of DNA in spermatozoa is half that in 2n hepatocytes, but by the conventional Hoe staining procedure the fluorescence intensity of spermatozoa is higher than that of 2n hepatocytes, due to differences in accessibility of the dye to DNA. In contrast, by the NaCl-aided procedure, the fluorescence intensity of 2n hepatocytes was twice that of spermatozoa. The effectiveness of the NaCl-aided Hoe staining method was checked using cultivated human gingival cells and hepatocytes of LEC rats with hereditary hepatitis. In all cases, reasonable proportionality between the fluorescence intensity and the amount of DNA was observed.     

Decrease of DNA per cell during development of the lens in chickens

Summary Developmental changes in the amount and conformation of DNA in chicken lens were studied. For this, DNA in situ in lens fiber cell nuclei of chickens was examined by microfluorometry with Hoechst 33258 (Hoe) fluorochrome. On 1 M NaCl-aided Hoe staining, by which the amount of DNA can be determined accurately, the fluorescence intensity of lens fiber cells was found to decrease with no change in that of the lens epithelial cells during development. On the contrary, on normal NaCl-free Hoe staining the fluorescence intensity of the lens cells was found to increase gradually during development. These results suggest that during development the amount of DNA in lens fiber cells decreases in association with some change in its conformation.     

Decrease of DNA per cell during development of the lens in chickens.

Developmental changes in the amount and conformation of DNA in chicken lens were studied. For this, DNA in situ in lens fiber cell nuclei of chickens was examined by microfluorometry with Hoechst 33258 (Hoe) fluorochrome. On 1 M NaCl-aided Hoe staining, by which the amount of DNA can be determined accurately, the fluorescence intensity of lens fiber cells was found to decrease with no change in that of the lens epithelial cells during development. On the contrary, on normal NaCl-free Hoe staining the fluorescence intensity of the lens cells was found to increase gradually during development. These results suggest that during development the amount of DNA in lens fiber cells decreases in association with some change in its conformation.     

Accurate determination of DNA content in single cell nuclei stained with Hoechst 33258 fluorochrome at high salt concentration

In an attempt to achieve accurate quantification of DNA levels in cell nuclei, we studied the influence of salt concentration on the fluorescence of cell nuclei complexed with Hoechst-33258 (Hoe) fluorochrome. The fluorescence of cell nuclei was compared with that of extracted DNA as well as that of nucleosome core. Conformational changes in these complexes were examined by measuring both fluorescence anisotropy and fluorescence lifetime in the nanosecond region. The results showed that the fluorescence of DNA-Hoe was quenched by the nucleosomal structure, there being an associated increase in anisotropy and a decrease in the fluorescence lifetime; however, the fluorescence was restored to the original level by the addition of a high concentration of NaCl, CsCl, or LiCl. The reduction in fluorescence may have been due to loss of fluorescence energy caused by collision of the fluorophore with histones in the nucleosome. The addition of 1 M NaCl to the medium used for staining with Hoe greatly stabilized the fluorescence of DNA in cell nuclei. The DNA content of individual cell nuclei was determined by comparing the fluorescence of these nuclei with that of a standard DNA solution. For lymphocytes and liver ploidy cells, reasonably accurate values were obtained by applying the present method.     

Accurate determination of DNA content in single cell nuclei stained with Hoechst 33258 fluorochrome at high salt concentration

Summary In an attempt to achieve accurate quantification of DNA levels in cell nuclie, we studied the influence of salt concentration on the fluorescence of cell nuclei complexed with Hoechst-33258 (Hoe) fluorochrome. The fluorescence of cell nuclei was compared with that of extracted DNA as well as that of nucleosome core. Conformational changes in these complexes were examined by measuring both fluorescence anisotropy and fluorescence lifetime in the nanosecond region. The results showed that the fluorescence of DNA-Hoe was quenched by the nucleosomal structure, there being an associated increase in anisotropy and a decrease in the fluorescence lifetime; however, the fluorescence was restored to the orginal level by the addition of a high concentration of NaCl, CsCl, or LiCl. The reduction in fluorescence may have been due to loss of fluorescence energy caused by collision of the fluorophore with histones in the nucleosome. The addition of 1 M NaCl to the medium used for staining with Hoe greatly stabilized the fluorescence of DNA in cell nuclei. The DNA content of individual cell nuclei was determined by comparing the fluorescence of these nuclei with that of a standard DNA solution. For lymphocytes and liver ploidy cells, reasonably accurate values were obtained by applying the present method.     

Parameters influencing the flow cytometric analysis of DNA sensitivity to nuclease S1

Some parameters that influence the analysis in situ of DNA sensitivity to digestion with nuclease S1 have been studied in isolated HeLa nuclei with flow cytometry. DNA staining with the intercalating fluorochrome propidium iodide allowed the nucleolytic activity on double-stranded (ds) DNA to be determined by monitoring the relative reduction in nuclear fluorescence intensity. Nuclei isolated in buffer at low ionic strength in order to decondense chromatin fibres, showed a lower fluorescence intensity than nuclei with native chromatin, after digestion with nuclease S1 under identical conditions. Nuclei prepared with dispersed chromatin and digested with increasing amounts of enzyme showed a decrease in fluorescence intensity that reached a limit value at about 50% of the value of undigested control samples. On the other hand, in nuclei with native chromatin, fluorescence intensity decreased only about 18%. The NaCl concentration in the reaction buffer strongly influenced the DNA sensitivity to S1 nuclease. By increasing salt molarity from 5 mM to 200 mM, the digestion of dsDNA was significantly reduced as also shown by the amount of released nucleotides from purified calf thymus DNA. The detection of DNA sensitivity to nuclease S1, as assessed by the cytometric method, was shown to be more sensitive than a biochemical technique involving hydrolysis of purines. These results indicate that both the procedure for nuclei isolation and the digestion conditions have to be carefully controlled when evaluating in situ the presence of S1-sensitive sites.     

Parameters influencing the flow cytometric analysis of DNA sensitivity to nuclease S1

Summary Some parameters that influence the analysis in situ of DNA sensitivity to digestion with nuclease S1 have been studied in isolated HeLa nuclei with flow cytometry. DNA staining with the intercalating fluorochrome propidium iodide allowed the nucleolytic activity on double-stranded (ds) DNA to be determined by monitoring the relative reduction in nuclear fluorescence intensity. Nuclei isolated in buffer at low ionic strength in order to decondense chromatin fibres, showed a lower fluorescence intensity than nuclei with native chromatin, after digestion with nuclease S1 under identical conditions. Nuclei prepared with dispersed chromatin and digested with increasing amounts of enzyme showed a decrease in fluorescence intensity that reached a limit value at about 50% of the value of undigested control samples. On the other hand, in nuclei with native chromatin, fluorescence intensity decreased only about 18%. The NaCl concentration in the reaction buffer strongly influenced the DNA sensitivity to S1 nuclease. By increasing salt molarity from 5 mM to 200 mM, the digestion of dsDNA was significantly reduced as also shown by the amount of released nucleotides from purified calf thymus DNA. The detection of DNA sensitivity to nuclease S1, as assessed by the cytometric method, was shown to be more sensitive than a biochemical technique involving hydrolysis of purines. These results indicate that both the procedure for nuclei isolation and the digestion conditions have to be carefully controlled when evaluating in situ the presence of S1-sensitive sites.     

Improvement of flow cytometry analysis and sorting of bull spermatozoa by optical monitoring of cell orientation as evaluated by DNA specific probing.

Flow cytometry is a potential method for the separation of X and Y bearing spermatozoa, on the basis of their relative DNA content evaluated by the fluorescence emission intensity due to specific fluorochrome DNA staining. However, spermatozoa DNA is highly condensed and nuclei exhibit flat non spherical shape, which can produce artefacts impeding accurate analysis. In order to avoid these limitations, decondensation of DNA performed by enzymatic treatment and a modification of the flow cytometer that orients the spermatozoa relative to the laser beam are generally used. In this work, we describe alternative methods and materials for selection of 1) decondensed and thus dead spermatozoa without orientation, sorted on the basis of only the 10% spermatozoa containing the least DNA (expected Y) and the 10% spermatozoa containing the more DNA (expected X), or 2) native spermatozoa homogeneously oriented using a simultaneous measurement of Axial light loss (extinction) and Forward angle light scatter. For testing enrichment of each selected fraction we have worked out a molecular hybridization procedure using X and Y specific DNA probes. We analyse and sort bull spermatozoa on these basis: the purity obtained for these fractions is 80% without orientation after enzymatic treatment, and 70% on live spermatozoa "optically" oriented.     

Immunofluorescent localization of lysine-rich histones in isolated nuclei from adult and embryonic chicken erythrocytes

Isolated nuclei from adult chicken erythrocytes were stained by indirect immunofluorescence for histones H5 and H1. Nuclei in 0.15 M NaCl stained for H5 showed internuclear variations in intensity of fluorescence from bright to dim. Most individual nuclei were homogeneously stained although some showed a bright rim around a dimmer interior. Treatment of nuclei with Tween 80 in 0.15 or 0.03 M NaCl also gave internuclear variation in intensity. Adult nuclei stained for H1 (in 0.15 or 0.03 M NaCl) showed little internuclear variation; most nuclei stained brightly with a brighter rim. Simultaneous staining of H5 and H1 in the same nuclei confirmed the variable fluorescence of H5 and consistent fluorescence of H1. Most nuclei showed the presence of both histones. Nuclei from embryonic blood cells also showed considerable internuclear variation of H5 fluorescence and less variation with H1 staining. For both histones the proportion of brightly staining nuclei increased with embryonic development. Difficulties in interpreting quantitative variations in immunofluorescence are discussed.     

Propidium iodide staining correlates with the extent of DNA degradation in isolated nuclei.

Gradual degradation of internucleosomal DNA is a hallmark of apoptosis and can be simulated by incubating isolated thymocyte nuclei in the presence of 5 mM Mg2+ and 5 mM Ca2+ at 37 degrees C. Staining of nuclei with the DNA binding fluorescent dye propidium iodide (PI) showed that intensity of fluorescence correlated with the extent of DNA degradation. PI fluorescence was increased in the presence of DNase I. Thus it seems that the cleavage of chromatin DNA by DNase 1 or by the endogenous enzyme increases the accessibility of DNA for the dye. No increase of fluorescence was observed in the presence of the known inhibitors of the endogenous endonuclease: Zn2+ and EGTA. However, the presence of Zn2+ led to decreased staining of the nuclei by PI and caused a shift in the scatter profile of the nuclei, suggesting that a conformational change of chromatin is induced by this ion. This correlation between intensity of PI staining and DNA degradation should be useful to compare endogenous nuclease levels in lymphocyte populations.     

Nucleus of the boar spermatozoon: structure and modifications in frozen, frozen-thawed, or sodium dodecyl sulfate-treated cells

After cryosubstitution and Epon embedding, or after Nanoplast embedding and very thin sectioning, the chromatin of ejaculated or diluted boar spermatozoa appears to be formed of DNA fibers embedded in a quite homogeneous matrix. After sodium dodecyl sulfate (SDS) treatment, and to a lesser extent after freeze-thawing, the DNA fibers are present mostly between cords, probably proteinaceous in nature. The quantity of free sulfhydryl (SH) groups, as calculated from staining by DACM and flow fluorometry, is increased in thawed or SDS-treated cells. The quantity of NH2 groups, calculated from electron microscopy image analysis of alcoholic phosphotungstic acid-stained cells, is decreased in thawed nuclei. The DNA is more accessible to the fluorochrome ethidium bromide after freeze-thawing, and its sensitivity to HCl hydrolysis is modified, during the Feulgen-like staining procedure using acriflavine. The X-ray energy dispersive analysis of cryosections of nuclei indicates that the slight separation of DNA and nucleoproteins in freeze-thawed spermatozoa could result from a dramatic modification of the nuclear ionic environment during thawing.     

Morphological and ultrastructural characterization of mammalian spermatozoa processed for flow cytometric DNA analyses

The morphological and ultrastructural changes that occur during preparation of porcine, bovine, and murine spermatozoa for flow cytometric quantification of the relative DNA content of the X- and Y-chromosome-bearing sperm populations were examined. Ejaculated spermatozoa from the boar and bull were washed using a series of dimethyl sulfoxide (DMSO) solutions prior to fixation, whereas the epididymal mouse spermatozoa were washed only in phosphate-buffered saline (PBS). Spermatozoa from all three species were then fixed in ethanol and processed for fluorochrome staining by a treatment regimen consisting of sulfhydryl reduction and proteolysis. The processed sperm nuclei were stained for DNA with the fluorochrome, 4′-6-diamidino-2-phenylindole (DAPI) before quantification by flow cytometry. Scanning and transmission electron micrographs of sperm heads taken at various steps of the preparation and staining procedures show 1) that the rigorous washing procedure disrupted the plasma and outer acrosomal membranes, 2) that ethanol fixation resulted in removal of the outer membranes and disintegration of the nuclear envelope, and 3) that thiol and proteolysis treatment removed the remaining cellular organelles including the tail and rapidly induced partial decondensation of the tightly packed chromatin. Sequential micrographs showed that the nuclear matrix of all three species increased in thickness about twofold during the preparation and staining. Consequently, the harsh procedures currently used for quantitative staining of DNA for high-resolution flow cytometric analyses destroy most cellular organelles and thereby prevent simultaneous characterization of DNA content and other sperm cell constituents.     

Mitochondrial Membrane Potential and DNA Stainability in Human Sperm Cells: A Flow Cytometry Analysis with Implications for Male Infertility

Sperm cells from control donors of proven fertility and men from barren couples were studied by conventional procedures, i.e., light microscopy as well as flow cytometry. Light microscopy analysis of semen included the measurement of spermatozoa concentration, morphology, and motility. All the men from barren couples were asthenozoospermic at the conventional analysis of semen samples. Flow cytometry was applied to study two important parameters of sperm cells: mitochondrial membrane potential (MMP) assessed by the cationic dye JC-1 and DNA stainability with propidium iodide (PI). JC-1 staining was more reliable than the classical procedure used for this purpose, i.e., rhodamine 123 (Rh123) staining, and allowed us to show a positive correlation between MMP and spermatozoa motility. Regarding DNA analysis, a higher relative percentage of immature spermatozoa, showing a high accessibility of DNA to the intercalating PI fluorochrome, was found in men from barren couples compared to donors of proven fertility. The relative percentage of immature spermatozoa was significantly higher in semen from oligoasthenozoospermic subjects. Moreover, a positive correlation was found between immature spermatozoa, as evaluated by PI staining, and cells with depolarized mitochondria, as evaluated by JC-1 staining, suggesting that spermatozoa defective for nuclear maturity could be functionally defective cells. No correlation between immature spermatozoa determined by FCM and immature spermatozoa determined by light microscopy was found, suggesting that these two techniques assess sperm cell maturity at different levels.     

Cytofluorometric Determination of Nuclear DNA in Living and Preserved Algae

Three DNA-localizing fluorochromes used in conjunction with epi (incident) UV illumination were examined for sensitivity and selectivity for the cytofluorometric determination of nuclear DNA in ten species of six algal genera: Mougeotia, Oedogonium, Sirogonium, Spirogyra and Zygnema among the green algae, and the marine red alga Polysiphonia boldii. In comparison with absorption photometry for the determination of nuclear DNA, the cytofluorometric procedure proved to be simpler and considerably more sensitive. Following staining with 4',6-diamidino-2-phenylindole (DAPI), nuclei fluoresce blue-white, the fluorescence intensity of the DNA-DAPI complex being considerably greater than that of the unbound dye molecule. Algal strains stained with 2,5-bis[4'-aminopheny](1')]-1,3,4-oxadiazole (BAO) also showed brilliant blue-white nuclear fluorescence. Although the BAO schedule requires the use of freshly prepared dye and sulfite water, and careful control of hydrolysis, nuclear fluorescence of the stained specimens does not fade under irradiation of the UV beam as rapidly as it does with certain other fluorochrome procedures. A more useful fluorochrome was the fungal antibiotic mithramycin. Its staining schedule is simple and the bright orange-yellow fluorescence of the nuclei is associated with an exceptional degree of sensitivity and specificity for DNA. Forty-eight-year-old preserved filaments of Spirogyra jatobae, stained with either BAO or mithramycin, exhibited a fluorescence brilliance of nuclear and chloroplast DNA equal to that of fresh specimens of this species. The three schedules, but particularly the one with mithramycin, have proven useful in providing indirect evidence for variation in ploidy level in several of the above algal genera, and in verifying the assumed ploidy level of the gametophyte (haploid) and tetrasporophyte (diploid) of Polysiphonia boldii     

Flow cytometry of DNA in mouse sperm and testis nuclei.

Mutations that occur in spermatogenic cells may be expressed as changes in DNA content, but developmentally-dependent alteration of its staining properties complicates the quantitation fo DNA in individual germ cells. These alterations have been studied with flow cytometric techniques. Nuclei from mouse testis cells and sperm were stained by the acriflavine-Feulgen method. The fluorescence intensity frequency distribution of nuclei of testis cells was characterized by 2 major and 5 minor peaks. Nuclei sorted from the various peaks with a fluorescence-activated cell sorter were identified microscopically. These data were confirmed by generation of peaks with nuclei prepared from cell suspensions enriched in specific cell types. One of the major peaks corresponded to round spermatid nuclei. The other major peak, located at 0.6 of the fluorescence intensity of the round nuclei, corresponded to elongated spermatid nuclei. Purified nuclei of epididymal and vas deferens spermatozoa displayed asymmetric fluorescence distributions. A minor peak at 0.8 the intensity of the round spermatid nuclei was tentatively assigned to elongating spermatids. 2 of the minor peaks, located at 1.7 and 2.0 times the fluorescence intensity of the round nuclei, corresponded to clumps of 2 haploid and diploid nuclei. The additional peaks, located at 3.0 and 3.7 times the fluorescence intensity of round spermatid nuclei correspond to leptotene and zygotene spermatocytes and to late pachytene spermatocytes, respectively. These peaks contained clumps of nuclei. The homogeneity of the nuclei sorted from the peaks, as well as the relative sizes of the peaks, was enhanced when the nuclei were prepared from cells enriched in specific stages of development. The relative fluorescence intensities of the various testis nuclei were characteristic and repeatedly found but were not stoichiometric with the DNA content of the nuclei.     

Are seeds suitable for flow cytometric estimation of plant genome size?

BACKGROUND: Nuclear DNA content in plants is commonly estimated using flow cytometry (FCM). Plant material suitable for FCM measurement should contain the majority of its cells arrested in the G0/G1 phase of the cell cycle. Usually young, rapidly growing leaves are used for analysis. However, in some cases seeds would be more convenient because they can be easily transported and analyzed without the delays and additional costs required to raise seedlings. Using seeds would be particularly suitable for species that contain leaf cytosol compounds affecting fluorochrome accessibility to the DNA. Therefore, the usefulness of seeds or their specific tissues for FCM genome size estimation was investigated, and the results are presented here. METHODS: The genome size of six plant species was determined by FCM using intercalating fluorochrome propidium iodide for staining isolated nuclei. Young leaves and different seed tissues were used as experimental material. Pisum sativum cv. Set (2C = 9.11 pg) was used as an internal standard. For isolation of nuclei from species containing compounds that interfere with propidium iodide intercalation and/or fluorescence, buffers were used supplemented with reductants. RESULTS: For Anethum graveolens, Beta vulgaris, and Zea mays, cytometrically estimated genome size was the same in seeds and leaves. For Helianthus annuus, different values for DNA amounts in seeds and in leaves were obtained when using all but one of four nuclei isolation buffers. For Brassica napus var. oleifera, none of the applied nuclei isolation buffers eliminated differences in genome size determined in the seeds and leaves. CONCLUSIONS: The genome size of species that do not contain compounds that influence fluorochrome accessibility appears to be the same when estimated using specific seed tissues and young leaves. Seeds can be more suitable than leaves, especially for species containing staining inhibitors in the leaf cytosol. Thus, use of seeds for FCM nuclear DNA content estimation is recommended, although for some species a specific seed tissue (usually the radicle) should be used. Protocols for preparation of samples from endospermic and endospermless seeds have been developed.     

A flow cytometric study of DNA staining in situ in exponentially growing and stationary Euglena gracilis

DNA stainability by different fluorochromes has been compared in exponentially dividing and stationary Euglena cells. With the intercalating fluorochromes, ethidium bromide, acridine orange and DAPI, a decrease of fluorescence intensity of the G1 cells is observed when cells enter stationary stage. However this decrease of fluorescence is not obtained with the nonintercalating fluorochrome Hoechst 33258. If nuclear basic proteins are extracted, however, the intensity of staining by either Hoechst 33258 or ethidium-bromide is comparable in stationary and dividing cells. Therefore, the decrease of fluorescence intensity of the G1 cells observed during the transition from exponential to stationary phase is not due to a loss of DNA but is related to the exposure of chromatin binding sites for ethidium bromide. In Euglena cells, DNA accessibility for intercalating fluorochromes depends upon chromatin structure and consequently upon cell age.     

Unstainable DNA in cell nuclei. Comparison of ten different fluorochromes

Ten fluorochromes with specificity for DNA were used to compare the stainability of nuclei of exponentially growing, nondifferentiated Friend leukemia (FL) cells with that of dimethylsulfoxide-induced, fully differentiated FL cell nuclei. Decreased accessibility of DNA to several dyes, particularly pronounced in the case of some intercalators, was observed in differentiated cells. Dye binding was also compared for both sets of nuclei following extraction of nuclear proteins, mostly histones, with 0.1-N HCl. Acid extraction of nuclear proteins increased the accessibility of DNA to varying degrees, depending upon the fluorochrome. In most cases, the differences in fluorescence between differentiated and nondifferentiated nuclei stained with most intercalating dyes was abolished by acid treatment. The results are discussed in terms of the mode of interaction between DNA and the various fluorochromes and the factors associated with chromatin structure, which may affect or be associated with different degrees of proliferative activity.     

Increased variability in nuclear DNA content of testis cells and spermatozoa from mice with irregular meiotic segregation.

Variability in DNA content to testis cells and sperm from F₁ hybrids between the laboratory mouse (M. muscullus) and the tobacco mouse (M. poschiavinus), has been determined by flow cytometry (FMC). The F₁ hybrid mouse is known to be heterozygous for seven metacentric chromosomes produced by Robertsonian fusion. Enriched populations of nuclei from late pachytene spermatocytes and round spermatids were obtained by velocity sedimentation. These nuclei, as well as epididymal sperm nuclei and spleen cells, were stained by the acriflavin-Feulgen technique for DNA and measured by FCM. Peaks in the fluorescence intensity frequency distributions resulting from these measurements were analyzed to determine their mean fluorescence intensities and their widths (coefficients of variation). Because mean intensities of corresponding cell types from M. musculus and the F₁ hybrids were identical, the average DNA contents were taken to be the same. The average coefficients of variation of the peaks to fluorescence from the pachytene, spermatid, and sperm nuclei and spleen cells from M. muscullus animals were about 5%. While the peaks of fluorescence from spleen cells and pachytene nuclei from f₁ hybrids also had average coefficients of variation of 5%, post-meiotic nuclei from spermatids and spermatozoa had coefficients of variationof 8%. From these results we conclude that, in these F₁ hybrids, abnormal meiotic segregation causes an increased variability of 6% in the amount of DNA in the spermatozoa.     

Plant DNA flow cytometry and estimation of nuclear genome size

BACKGROUND: DNA flow cytometry describes the use of flow cytometry for estimation of DNA quantity in cell nuclei. The method involves preparation of aqueous suspensions of intact nuclei whose DNA is stained using a DNA fluorochrome. The nuclei are classified according to their relative fluorescence intensity or DNA content. Because the sample preparation and analysis is convenient and rapid, DNA flow cytometry has become a popular method for ploidy screening, detection of mixoploidy and aneuploidy, cell cycle analysis, assessment of the degree of polysomaty, determination of reproductive pathway, and estimation of absolute DNA amount or genome size. While the former applications are relatively straightforward, estimation of absolute DNA amount requires special attention to possible errors in sample preparation and analysis. SCOPE: The article reviews current procedures for estimation of absolute DNA amounts in plants using flow cytometry, with special emphasis on preparation of nuclei suspensions, stoichiometric DNA staining and the use of DNA reference standards. In addition, methodological pitfalls encountered in estimation of intraspecific variation in genome size are discussed as well as problems linked to the use of DNA flow cytometry for fieldwork. CONCLUSIONS: Reliable estimation of absolute DNA amounts in plants using flow cytometry is not a trivial task. Although several well-proven protocols are available and some factors controlling the precision and reproducibility have been identified, several problems persist: (1) the need for fresh tissues complicates the transfer of samples from field to the laboratory and/or their storage; (2) the role of cytosolic compounds interfering with quantitative DNA staining is not well understood; and (3) the use of a set of internationally agreed DNA reference standards still remains an unrealized goal.