Histochemical study on the maturation of human megakaryocytes using microfluorometry.

A microcytofluorometrical DNA measurement was basically studied and was applied to single megakaryocytes previously identified on a Wright-Giemsa stained smear. The smear was first photographed and the location of each megakaryocyte was recorded on a cell map. The smear was then bleached with 50% acid ethanol and absolute methanol, and re-stained with 4',6-diamidino-2-phenylindole (DAPI) reagent (pH 7.4) at 4 degrees C. Nuclear blue fluorescence was observed and the intensity of this fluorescence was proportional to the amount of DNA with the coefficient of variation (CV) of 3.6% when stained for 30 min. After 30 min DAPI staining, the DNA measurement was microcytofluorometrically performed in single megakaryocytes which had been morphologically classified into 4 groups on the basis of cytoplasmic maturation, Bessis' classification, assessed on Wright-Giemsa-stained bone-marrow smears from normal human beings. The histograms of the cells did not show any difference in DNA ploidy distribution among the classes: that is, the DNA histograms disclosed ploidy distribution from 4 N to 64 N with the largest population of 16 N. These findings suggest that nuclear DNA synthesis is completed before platelet production starts. This method is useful for comparing the morphological features and DNA content of single megakaryocytes.     

Comparison of the DNA content of megakaryocytes identified immunologically with that identified morphologically

 We devised a new microfluorometric method for determining the ploidy of megakaryocytes identified immunologically in bone marrow smears. The smears were immunostained by incubation with mouse monoclonal anti-glycoproteins (GP) IIb antibodies, followed by fluorescein isothiocyanate-conjugated goat anti-mouse IgG antibodies. They were then stained with 4′,6-diamidino-2-phenylindole (DAPI). Megakaryocytes were identified by their GPIIb immunofluorescence using a microfluorometer and, after the filters were changed, their DNA content was assayed by measuring the intensity of DAPI fluorescence. This intensity was shown to be proportional to the DNA content when the aperture of the objective lens was reduced. We compared these results with those obtained when megakaryocytes were identified morphologically, using DAPI staining after Wright-Giemsa destaining. In all 12 normal controls, the ploidy peaks were shown to be 16N by both methods, and the mean ploidy detected by the immunological method was only reduced 0.961 times relative to the estimate from the morphological method. In contrast, in eight myelodysplastic syndrome (MDS) patients, the ploidy peaks were either 8N or 4N and the mean was reduced by 0.906 times (P=0.018). Thus we could immunologically identify small megakaryocytes which we could not identify morphologically. Therefore, this method is useful for measuring megakaryocytic ploidy, especially in the pathological megakaryocytes of MDS patients. Accepted: 29 April 1997     

Bivariate cytofluorimetric analysis of DNA and nuclear protein content in plant tissue

Summary Techniques of static biparametric cytofluorimetry were developed to measure DNA and protein fluorescence simultaneously in the same nucleus stained with 4,6-diamidino-2-phenylindole (DAPI) and fluorescein isothiocyanate (FITC) fluorochromes. With these cytofluorimetric procedures, we analysed DNA and nuclear protein content in root apices during the first 72 h of pea seed germination. This method allows a more reliable, rapid and less expensive measurement of DNA and proteins than cytophotometry. Nuclear protein content can be considered as a second parameter to define subcompartments of cell cycle phases; it offers the possibility of studying the progression of plant cells through cell cycle and its control in greater detail.Abbreviations DAPI 4,6-diamidino-2-phenylindole - FITC fluorescein isothiocyanate - PI propidium iodide - Tris Tris(hydroxymethyl) aminomethane     

Reverse fluorescent chromosome banding with chromomycin and DAPI

Two DNA binding guanine-specific antibiotics, chromomycin A₃ (CMA) and the closely related mithramycin (MM), were used as chromosome fluorescent dyes. Root-tip metaphase chromosomes of three plant species and human metaphase chromosomes were sequentially stained with CMA or MM and the DNA binding AT-specific fluorochrome 4-6-diamidino-2-phenylindole (DAPI). In some cases a non-fluorescent counterstain was used as contrasting agent: methyl green in conjunction with CMA, and actinomycin D (AMD) in combination with DAPI. — In all three plant species, Vicia faba, Scilla siberica, and Ornithogalum caudatum, the nucleolus organiser regions and/or associated heterochromatin displayed very bright fluorescence with CMA and MM and, in general, heterochromatic segments (C-bands) which were bright with CMA and MM were pale with DAPI whereas segments which were dim with CMA and MM displayed very bright fluorescence with DAPI. — Human metaphase chromosomes showed a small longitudinal differentiation in CMA fluorescence, which was essentially the reverse of the banding pattern obtained with AMD/DAPI double-staining, but of lower contrast. The CMA-banding pattern appears to be similar to the pattern found by R-banding procedures.     

Optical studies of the interaction of 4′-6-diamidino-2-phenylindole with DNA and metaphase chromosomes

The optical absorption and fluorescence characteristics of 4-6-diamidino-2-phenylindole (DAPI) with DNA and chromosomes were studied. There is a decrease in extinction coefficient and shift in the absorption spectra to a higher wavelength when the dye binds to DNA. The fluorescence of DAPI is enhanced by both A-T and G-C base-pairs. The enhancement by A-T rich is significantly greater than by G-C rich DNA. The dye produces a localized bright fluorescence in centromeric regions of mouse chromosomes and the constrictions of human chromosomes 1 and 16; these regions are known to contain A-T rich DNA and show dull fluorescence when treated with quinacrine. This dye may be useful for identifying A-T rich region in chromosomes. The fluorescence of DAPI bound to polynucleotides or chromosomes is partially quenched by the introduction of BrdU. This suppression of dye fluorescence allows optical detection of sister chromatid exchanges and chromosome region containing DNA with an unequal distribution of thymidine between polynucleotide chains after BrdU incorporation.     

Measurement of DNA content in single cells morphologically identified on smears

A method was developed for measuring the nuclear DNA content in single cells previously identified on a bone marrow smear stained by the Wright-Giemsa method. The smear was first photographed and the location of individual cells, identified by morphology, was recorded on a cell map. The smear was then bleached with 50% acid ethanol and absolute methanol, and re-stained by the Feulgen method in 0.05% pararosaniline Schiff's reagent (pH 2.3) at 7 degrees C for 10 min. Nuclear red fluorescence was observed and the intensity of this fluorescence was proportional to the amount of DNA after prior irradiation of smears with green light for 9 hr. The method is useful for measuring cell DNA content in heterogeneous cell populations when morphological cell identification is required.     

Standardization of the post-irradiation method to eliminate primary fluorescence in cytofluorometry.

Thorough irradiation of specimen with the strong excitation light after fluorescent Feulgen staining destroys the primary fluorescence in the background with stabilization of specific fluorescence of pararosaniline (post-irradiation method). An apparatus to perform effective post-irradiation was developed and irradiation condition for DNA cytofluorometry on a pararosaniline Feulgen stained smear was standardized. After 10 h irradiation on this condition, the primary fluorescente in the background is almost completely cleared away. Proportionality between amount of fluorescence and DNA content is perfectly preserved and standard deviation in each class of ploidy becomes to be less than 5% of the mean DNA value. The standardized post-irradiation enables us to obtain reproducibly the same values of fluorescence on Feulgen stained nuclei of the same cell population in different smears even if the staining conditions in Schiff's solution might be different to some extent.     

"Liquidless" cell staining by dye diffusion from gels and analysis by laser scanning cytometry: potential application at microgravity conditions in space

BACKGROUND: Conventional staining of cells or tissue sections on microscope slides involves immersing the slides into solutions of dyes then rinsing to remove the unbound dye. There are instances, however, when use of stain solutions is undesirable-e.g., at microgravity conditions in space, where the possibility of accidental spill (many dyes are known carcinogens) introduces health hazard. Likewise, transporting bulk of liquid stains and rinses may be burdensome in certain situations such as field expeditions or combat. METHODS: The "liquidless" staining procedure is proposed in which the dyes are contained in thin strips of hydrated polyacrylamide or gelatin gels that have been presoaked in the stain solutions. Fluorochromes that have affinity to DNA (propidium iodide, PI; 4,6-diamidino-2-phenylindole, DAPI, Hoechst 33342) or to protein (sulforhodamine 101) were used to saturate the gels. The gel strips were placed over the prefixed cells or tissue sections deposited on microscope slides and relatively low (20 g/cm2) pressure was applied to ensure the contact. The cells were also stained by using commercially available mounting media into which DAPI or PI were admixed. Intensity of fluorescence of the PI stained cells was measured by laser scanning cytometry (LSC). RESULTS: Satisfactory cell and tissue staining, with minimal background, was achieved after 10-20 min contact between the cells and gels. Optimal concentrations of the dyes in the solutions used to presoak the gels was found to be 2-4-fold higher than the concentrations used routinely in cytometry. The measurements of intensity of cellular fluorescence by LSC revealed that the staining of DNA was stoichiometric as reflected by the characteristic cellular DNA content frequency histograms with distinct G1, S, and G2/M cell populations and 2:1 ratio of G2/M to G1 peak fluorescence. Individual gels can be saturated with more than a single dye-e.g., to obtain differential DNA and protein staining. Cell staining with DAPI or PI in the gelatin-based mounting media led to high fluorescence background while staining with DAPI in "aqueous" medium was satisfactory. CONCLUSIONS: Relatively fast staining of cells or tissue sections on microscope slides can be achieved by nonconvective dye diffusion using hydrated gels permeated with the dyes, applied to cells at low pressure. The quality of the staining provided by this methodology is comparable to conventional cell staining in dye solutions.     

Standardization of the post-irradiation method to eliminate primary fluorescence in cytofluorometry

Summary Thorough irradiation of specimen with the strong excitation light after fluorescent Feulgen staining destroys the primary fluorescence in the background with stabilization of specific fluorescence of pararosaniline (post-irradiation method).An apparatus to perform effective post-irradiation was developed and irradiation condition for DNA cytofluorometry on a pararosaniline Feulgen stained smear was standardized. After 10 h irradiation on this condition, the primary fluorescence in the background is almost completely cleared away. Proportionality between amount of fluorescence and DNA content is perfectly preserved and standard deviation in each class of ploidy becomes to be less than 5% of the mean DNA value.The standardized post-irradiation enables us to obtain reproducibly the same values of fluorescence on Feulgen stained nuclei of the same cell population in different smears even if the staining conditions in Schiff's solution might be different to some extent.Partly supported by Alexander von Humboldt-Stiftung     

Analysis of megakaryocyte ploidy in patients with thrombocytosis

The DNA content of bone marrow megakaryocytes was analyzed in 24 patients with myeloproliferative disorders, 23 patients with secondary thrombocytosis and 15 normal volunteers using 2-color flow cytometry. Compared with normal controls, the majority of patients with secondary thrombocytosis, polycythemia vera and essential thrombocytosis exhibited a relative increase in higher ploidy (greater than 16N) cells. In contrast, patients with chronic myelogenous leukemia exhibited an increase in lower ploidy cells (less than 16N), with a modal DNA content of 8N. Patients with myeloproliferative disorders tended to show a decrease in the 16N megakaryocyte population compared with patients with secondary thrombocytosis. No correlation between ploidy distribution and platelet count was observed.     

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     

NASA/American Cancer Society High-Resolution Flow Cytometry Project - II. Effect of pH and DAPI concentration on dual parametric analysis of DNA/DAPI fluorescence and electronic nuclear volume

BACKGROUND: In the present paper, we describe the effect of 4', 6-diamidino-2-phenylindole (DAPI) dihydrochloride concentration and pH on the resolution of DNA distribution histograms generated by dual-parametric simultaneous analysis of DNA content and electronic nuclear volume (ENV). METHODS: Nuclei from tissue culture cell lines and frozen human solid tumors were isolated in nuclear isolation media containing different concentrations of DAPI, at various pH levels, and analyzed on a NASA/American Cancer Society (ACS) flow cytometer. Samples stained with propidium iodide/hypotonic citrate and analyzed in a Coulter XL flow cytometer were used for comparison. RESULTS: Nuclei stained with DAPI concentration of 1-3 microg/ml, pH 6.0, gave the best resolution for the detection of the near-diploid and near-tetraploid populations. Simultaneous use of ENV and DAPI/DNA fluorescence under these conditions identified subpopulations that otherwise could not be detected by DNA analysis alone. CONCLUSIONS: Staining at 1-3 microg/ml DAPI, pH 6.0, was optimal for the detection of aneuploid populations, especially the near-diploid and/or near-tetraploid populations in human tumors.     

Determination of ploidy level and nuclear DNA content in blueberry by flow cytometry

The technique of DNA flow cytometry was used to study variation in DNA content among different ploidy levels, as well as among diploid species, of Vaccinium section Cyanococcus. In a sample of plants of varying ploidy level, the relative fluorescence intensity (RFI) of nuclei stained with propidium iodide was a function of the number of chromosome sets (x), as represented by the linear equation RFI=3.7x-2.3 (r²=95%). The data indicated that DNA flow cytometry could be useful for the determination of ploidy level at the seedling stage in blueberry. They also suggest that conventional polyploid evolution has occurred in this section of the genus Vaccinium with an increase in nuclear DNA content concurrent with the increase in chromosome number. The nuclear DNA content of diploid species of Vaccinium section Cyanococcus was estimated from the relationship of the observed RFI to an internal known DNA standard (trout red blood cells). A nested analysis of variance indicated significant variation among species, as well as among populations within species, in nuclear DNA content, although this variation was small compared to the variation among ploidy levels. The variation in nuclear DNA content corresponded to the phylogenetic relationships among species determined from previous studies.     

Correlation of grade of urothelial cell carcinomas and DNA histogram features assessed by flow cytometry and automated image cytometry.

OBJECTIVE: To analyse how DNA ploidy and S-phase fraction (SPF) by flow cytometry (FCM) and an optimised fully automatic DNA image cytometer (ICM) correlate with grade in TaT1 urothelial cell carcinomas (UC) of the urinary bladder. MATERIALS AND METHODS: Two-hundred-and twenty-eight consensus cases were analysed. Single cell suspensions were stained (DAPI for FCM, Feulgen for ICM). There was enough material for both FCM and ICM in 202 of these cases. FCM and optimised ICM measurements were performed on the 202 UCs. To discriminate between different grades, single- and multivariate analyses was performed on DNA histogram features obtained with the MultiCycle program (using DNA index (DI) and SPF). RESULTS: Overall measurement time of the adapted ICM method was 10.7 minutes per case (range 5.9-29.8 min.) and required little additional interactive object rejection (average 152 objects (84-298) on 3000 objects per case measured, which took 9.9 minutes on average, range 8.3-15.5 minutes). The ICM histograms looked much "cleaner" with less noise than the FCM graphs. The coefficient of variation (CV) of the diploid peak for ICM (5.4%) was significantly lower than for FCM (5.9%) (p<0.0001). ICM features were more strongly correlated to grade than FCM features. In multivariate analysis, the best discriminating set of features was DNA ploidy and SPF (both by ICM). CONCLUSIONS: The adapted fully automated DNA ICM works very well for UCs. Low CV DNA ICM histograms are obtained in a time comparable to FCM. The DNA ICM results have stronger discriminative power than DNA FCM for grade in TaT1 UCs.     

Satellite DNA and cytological staining patterns in heterochromatic inversions of human chromosome 9

Summary A series of partial inversions of the heterochromatic C-band of chromosome 9 have been stained with distamycin A plus 4,6-diamidino-2-phenyl-indol-2 HCl (DA/DAPI) and found to consist of three classes: (a) those in which only the C-band in the long arm fluoresces with DA/DAPI (these are the most frequent), (b) those in which only the C-band in the short arm fluoresces with DA/DAPI, and (c) those in which the C-bands in both arms fluoresce with DA/DAPI.There are also differences in the satellite DNA content of each type of inversion as measured by hybridisation in situ. Types (a) and (b) have satellite DNA contents similar to those of their normal homologues, while type (c) has a satellite DNA content almost double that of the normal homologue.It appears that DA/DAPI specifically stains heterochromatin that contains satellite DNA.The ability to distinguish these three types of inversion may help to resolve the question of the clinical significance of such inversions.     

Production of first and second generations aposporous gametophytes fromPyrrosia piloselloides (L.) Price frond strips cultured in vitro

Summary Second generation aposporous gametophytes were obtained from sporophytes derived from first generation aposporous gametophytes, which in turn came from the mature fronds grown from spores in the laboratory. Murashige and Skoog modified medium in 1% agar supplemented with sugar alcohols (sorbitol, mannitol), auxins (NAA, 2,4-D) and cytokinin (BA) promoted a higher percentage of aposporous development from mature fronds ofPyrrosia piloselloides derived from aseptically cultured spores as compared with those obtained from plants in the field. A method using 46-diamidino-2-phenyl indole and fluorescence microscopy correlated the deoxyribonucleic acid contents of the aposporous gametophytes and sporophytes derived from them with their ploidy level.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DAPI 46-diamidino-2-phenyl indole - DNA deoxyribonucleic acid - MS Murashige and Skoog medium - BA benzyladenine - NAA 1-naphthaleneacetic acid     

Distribution of the Golgi apparatus in the mitosis of cultured tobacco cells as revealed by DiOC6 fluorescence microscopy

Summary We developed a new method for distinguishing the Golgi apparatus from the other membranous organelles which contain DNA, such as mitochondria and chloroplasts, under a fluorescence microscope. Thin sections of cells embedded in Technovit 8100 resin were stained with both 3,3-dihexyloxacarbocyanine iodide (DiOC₆) and 4,6-diamidino-3-phenylindole (DAPI), and those three membranous organelles were observed under an epifluorescence microscope. The Golgi apparatus, which do not contain DNA, were easily recognized when the two images stained with DiOC₆ and DAPI were superimposed using an image processor. Using this method, we investigated the dynamics of cellular membranes and organelles during the mitotic cycle of synchronized cultured tobacco cells BY-2 (Nicotiana tabacum L. cv. Bright Yellow 2). The Golgi apparatus did not accumulate in the rim of the formating early cell plate at anaphase, while it accumulated near the maturing cell plate at telophase, and this accumulation seemed to be related to the maturation of cell plates. To confirm this hypothesis, synchronized BY-2 cells were treated with caffeine, which is known to inhibit the cell plate formation. Most of the cells treated with caffeine remained in a phase in which Golgi vesicles were accumulated at the equatorial plate, but the cell plate was only partially maturing. The Golgi apparatus accumulated only near the partially maturing cell plate, but not by the equatorial plate where the Golgi vesicles had accumulated.Abbreviations DiOC₆ 3,3-dihexyloxacarbocyanine iodide - DAPI 4,6-diamidino-3-phenylindole - LSD a modified Linsmaier and Skoog's medium containing 2,4-D     

DNA is present in the nucleomorph of cryptomonads: Further evidence that the chloroplast evolved from a eukaryotic endosymbiont

Summary The nucleomorph is a unique self-replicating organelle which is invariably present in the periplastidal compartment of cryptomonads. The nucleomorph ofCryptomonas abbreviata is located in a groove on the inner face of the pyrenoid. When JB-4-embedded sections ofC. abbreviata are stained with 4-6-diamidino-2-phenylindole (DAPI), the nucleomorph exhibits a blue fluorescence characteristic of DNA-DAPI complexes. This fluorescence is removed by DNase digestion, but not by RNase. When cells are prepared for electron microscopy by the method of Ryter and Kellenberger (Schreil 1964), a network of fine DNA-like fibrils is observed in the nucleomorph matrix. It is estimated that the nucleomorph contains between 10⁸ and 10⁹ daltons of DNA. The presence of DNA in nucleomorphs strongly supports the hypothesis that the nucleomorph is the vestigial nucleus of a eukaryotic endosymbiont. It is postulated that this eukaryotic symbiont was an ancestral red alga or an organism closely related to red algae. The cryptomonad host cell, on the other hand, is not evolutionarily close to any other group of algae.     

Direct measurement of femtogram amounts of DNA in cells and chloroplasts by quantitative microspectrofluorometry

Absolute DNA amounts of individual chloroplasts were determined by measuring the fluorescence intensity of chloroplasts stained with 4',6-diamidino-2-phenylindole (DAPI) relative to that of the bacterium Pediococcus damnosus (cerevisiae) smeared on the same slide. An absolute DNA content of 7.7 X 10(15) g for a standard P. damnosus cell type was calculated by comparing the relative fluorescence values and frequency of each stage of cellular development in a culture to the average DNA content of all cell types determined by chemical methods. Chlorophyll was extracted from the chloroplasts during fixation so that chlorophyll autofluorescence was not present when DAPI fluorescence was measured. Absolute amounts of DNA could then be determined for single chloroplasts, either within cells that were individually selected from a mixed cell population or in small preparations of isolated chloroplasts. The DNA amounts of chloroplasts from mesophyll cells determined in this way were similar to the values previously determined by bulk averaging methods. Chloroplast DNA amounts from different cell types of the leaf could be measured by microspectrofluorometry, and it was found that chloroplasts from spinach epidermal cells contained about half as much DNA as chloroplasts from adjacent mesophyll cells.     

Fluorescence microscopy of DNA-protein structures from osmotically lysed mitochondria of yeast and tobacco

Summary The mitochondrial nucleoid is a compact structure composed of DNA and protein. By fluorescence microscopy, decondensation of the nucleoids was observed when yeast and tobacco mitochondria were osmotically lysed and subjected to an electric field. Structures stained with ethidium bromide were seen moving toward either the anode or the cathode. Since the movement of deproteinized DNA is toward the anode, the structures moving toward the cathode represent DNA-protein complexes with a net positive charge. Nucleoid decondensation and unfolding of the DNA probably resulted from the removal of weakly bound proteins; yet high-affinity basic proteins were evidently retained yielding cationic DNA-protein structures. Some of the positively charged structures were observed to break, presumably at single-stranded DNA regions, releasing negatively charged particles. The DNA-protein structures were complex branching forms larger than the unit genome, suggesting that multigenomic, concatemeric DNA is present within the mitochondria.Abbreviations DAPI 4,6-diamidino-2-phenylindole - EtBr ethidium bromide - HMG high-mobility group - mt-genome mitochondrial genome - mt-nucleoid mitochondrial nucleoid - PFGE pulsed-field gel electrophoresis - pt-nucleoid plastid nucleoid - ssDNA single-stranded DNA