家蚕拟浆细胞具有吞噬功能

目的：通过观察家蚕Bombyx mori吞噬细胞的微细结构,来确定拟绛色细胞是否也具有吞噬功能。方法：用荧光小球微量注射家蚕pnd pS品系的幼虫,经荧光染色剂丫啶橙和碘化丙啶染色循环血细胞后,在荧光显微镜下观察并扫描拍摄。结果：观察发现除颗粒细胞和浆血细胞外,一些原血球细胞（血干细胞）和拟绛色细胞（多酚氧化酶）也能吞噬荧光小球。在拟绛色细胞里还发现许多和颗粒细胞一样的能被丫啶橙染色的颗粒。尽管在小球细胞中没有发现被吞噬的荧光小球,但该类血球有比较多的能被丫啶橙染色的大颗粒,这表明它们可能是已经被吞噬的凋亡小体。结论：除颗粒细胞和浆血细胞外,一些原血球细胞和拟绛色细胞也能吞噬荧光小球。说明拟绛色细胞也具有吞噬功能。     

Hemocyte differentiation in the hematopoietic organs of the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>: prohemocytes have the function of phagocytosis

Hemocytes isolated from the larval hematopoietic organs of the silkworm were classified following staining with acridine orange and propidium iodide. Among the hemocytes isolated from the hematopoietic organs of whole fifth larval and wandering stages, most were prohemocytes (60%–70%) and oenocytoids (30%–40%). Granulocytes comprised only about 0.5%–1% at the wandering stage and were even rarer at other stages; no spherulocytes or plasmatocytes were found. Therefore, hemocyte differentiation inside larval hematopoietic organs is not as extensive as previously thought. Following 10–30 min in vitro culture of hemocytes isolated from larval hematopoietic organs, many young granulocytes and plasmatocytes appeared. Furthermore, during phagocytosis assays, prohemocytes were seen to adopt the morphology of plasmatocytes, containing fragments of phagocytosed cells. Our results underline the similarities between Drosophila and Bombyx hematopoiesis.     

Ultrastructural and functional characterization of circulating hemocytes from Plutella xylostella larva: Cell types and their role in phagocytosis

The hemocytes of different types encountered in the diamondback moth Plutella xylostella larvae of each instar and the development of the differential hemocytes counts were herein presented. Hemocytes classes/populations characterized based on their affinity with fluorescent dye (acridine orange) and ultrastructural differences comprised the prohemcoytes (<10–16%), plasmatocytes (22–65%), granulocytes (25–72%), oenocytoids (<1–9%), and spherulocytes (<1%). Prohemcoytes were the smallest cells with a comparatively tremendous nucleus. Plasmatocytes and granulocytes occupied the main proportion of total cell numbers. Oenocytoids were in a most stable presence, i.e. rotund in a diameter of 10 μm and with a nucleus deviated from the central location; however, sometimes with two nuclei which were adjoining with each other. Spherulocytes were rare and only could be observed occasionally. Ultrastructural investigation revealed that hemocytes in the diamondback moth larvae were of the typical model as in the Lepidoptera insect larvae. It is interesting to find that the cell which could phagocytize bacteria in vitro was granulocyte, not the other types of hemocytes, although plasmatocyte was usually declared to participate in this reaction in various previous studies.     

Immunocytochemical localization of β-1,3-glucan recognition protein in the silkworm,Bombyx mori

Summary A monospecific antibody against -1,3-glucan recognition protein (a 62 kDa protein) of the larval silkworm prophenoloxidase activating system was used to study the localization of the protein. Among tissues from 5th instar larvae, only hemocytes and plasma were shown to contain a 62 kDa polypeptide immunoreactive with the antibody. Ultra-thin sections of the hemocytes were stained by an indirect immunogold staining method. Labelling occurred in the granules and cytoplasm of granulocytes and in the spherules and cytoplasm of spherulocytes. It was most conspicuous in granules of granulocytes and uniformly labelled spherules of spherulocyte, whereas no labelling was evident in prohemocytes, plasmatocytes and oenocytoids. The results are discussed in relation to the mode of recognition of fungi as non-self in insect hemocoel.     

Automatic cell identification and enrichment in lung cancer. II. Acridine orange for cell sorting of sputum.

Fluorescence spectra were obtained from cells from sputum and pleural effusions stained with different fluorescent dyes and fixed by alternate methods. The spectra were referenced to a standard allowing for fluorescence comparisons of unstained and stained cells under various conditions. The metachromasia of acridine orange-stained cells offers nuclear/cytoplasmic differentiation in a single stain; mithramycin and propidium iodide do not. Unstained cells have an appreciable amount of green (546 nm) fluorescence, as does Carbowax in Saccomanno's preservative. Cytoplasm stained with acidine orange also has appreciable green fluorescence. Consequently, cells with much cytoplasm have high total fluorescence. Cytoplasmic fluorescence is negligible with mithramycin or propidium iodide. The metachromasia of acridine orange-stained cells is altered by alcohol and Carbowax levels in fixatives, keeping other factors constant.     

Acridine Orange—Methyl Green Fluorescent Staining of Nucleoli

The staining of nucleoli with the fluorescent dye acridine orange followed by counter-staining with methyl green differentially stained nucleoli in both plant and animal cells. The nucleoli fluoresced as bright structures highlighted against the quenched fluorescence of the chromatin. This technique provides a simple and highly reproducible method for differential staining of nucleoli.     

贝类血细胞硫代黄素T荧光染色方法

硫代黄素T( thioflavin T,TFT)是一种用于组织学的苯并噻唑荧光染料,因其对淀粉样蛋白有高亲和性而主要被用于淀粉样病变的荧光显微检测.本研究分别以软体动物门双壳纲的栉孔扇贝(Chlamys farreri)和中国蛤蜊(Mactra chinensis)、腹足纲的拟紫口玉螺(Natica janthosto...     

Fluorescent labeling of resin-embedded sections for correlative electron microscopy using tomography-based contrast enhancement

Locating areas of interest by electron microscopy can be laborious. This is particularly true for electron tomography, where the use of thicker sections may obscure relevant details in the projection images. We evaluated the applicability of fluorescent probes to thin plastic sections, in combination with fluorescence microscopy, as an aid in selecting areas for subsequent electron microscopic analysis. We show that pre-embedding labeling of DNA and RNA with acridine orange yielded a predominant nuclear stain. The stain greatly reduced the time needed to scan sections for mitotic cells, or cells with characteristic nuclei such as neutrophils. Post-embedding labeling with SYTOX green yielded a nuclear stain comparable to acridine orange, and wheat germ agglutinin (WGA) conjugated to Alexa Fluor 488 labeled mucous granules and the Golgi area in intestinal goblet cells. The fluorescent labels were visualized directly on sections on electron microscope grids. It was therefore possible to establish a coordinate system based on the position of the grid bars, allowing for easy retrieval of selected areas. Because the fluorescent probes were incompatible with osmium tetroxide treatment, contrast in the sections was faint. We propose a simplified electron tomography procedure for the generation of 2D views with enhanced contrast and resolution.     

Immune Defenses of the Invasive Apple Snail Pomacea canaliculata (Caenogastropoda,Ampullariidae): Phagocytic Hemocytes in the Circulation and the Kidney

Hemocytes in the circulation and kidney islets, as well as their phagocytic responses to microorganisms and fluorescent beads, have been studied in Pomacea canaliculata, using flow cytometry, light microscopy (including confocal laser scanning microscopy) and transmission electron microscopy (TEM). Three circulating hemocyte types (hyalinocytes, agranulocytes and granulocytes) were distinguished by phase contrast microscopy of living cells and after light and electron microscopy of fixed material. Also, three different populations of circulating hemocytes were separated by flow cytometry, which corresponded to the three hemocyte types. Hyalinocytes showed a low nucleus/cytoplasm ratio, and no apparent granules in stained material, but showed granules of moderate electron density under TEM (L granules) and at least some L granules appear acidic when labeled with LysoTracker Red. Both phagocytic and non-phagocytic hyalinocytes lose most (if not all) L granules when exposed to microorganisms in vitro. The phagosomes formed differed whether hyalinocytes were exposed to yeasts or to Gram positive or Gram negative bacteria. Agranulocytes showed a large nucleus/cytoplasm ratio and few or no granules. Granulocytes showed a low nucleus/cytoplasm ratio and numerous eosinophilic granules after staining. These granules are electron dense and rod-shaped under TEM (R granules). Granulocytes may show merging of R granules into gigantic ones, particularly when exposed to microorganisms. Fluorescent bead exposure of sorted hemocytes showed phagocytic activity in hyalinocytes, agranulocytes and granulocytes, but the phagocytic index was significantly higher in hyalinocytes.Extensive hemocyte aggregates (''islets'') occupy most renal hemocoelic spaces and hyalinocyte-like cells are the most frequent component in them. Presumptive glycogen deposits were observed in most hyalinocytes in renal islets (they also occur in the circulation but less frequently) and may mean that hyalinocytes participate in the storage and circulation of this compound. Injection of microorganisms in the foot results in phagocytosis by hemocytes in the islets, and the different phagosomes formed are similar to those in circulating hyalinocytes. Dispersed hemocytes were obtained after kidney collagenase digestion and cell sorting, and they were able to phagocytize fluorescent beads. A role for the kidney as an immune barrier is proposed for this snail.     

Phagocytosis, intracellular pH, and cell volume in the multifunctional analysis of granulocytes by flow cytometry

Phagocytosis of Escherichia coli K12 strain bacteria was used to measure by flow cytometry the functional activities of human granulocytes in whole blood or buffy coat preparations. In a first measurement, the increase in electric cell volume and acridine orange (AO) green and red fluorescence were used to quantify the degree of phagocytosis. In a second measurement, the intracellular pH and esterase activity of each cell were determined with 1,4-diacetoxy-2,3-dicyanobenzene to obtain information on the metabolic activities during phagocytosis and degradation of bacteria. The DNA of dead cells was simultaneously counterstained with propidium iodide in both assays. The volume, the AO green and red fluorescence, the internal pH, and esterase activity were automatically averaged for all granulocytes or lymphocytes of a measurement. The calculated mean values were transferred into the self-learning database of the DIAGNOS1-program system. The functional granulocyte parameters of normal healthy individuals can be used as reference values for the automated diagnosis of abnormal granulocytes in various infectious disease states. The assays require 1 ml of heparinized whole blood and the results are available within 1 hour.     

Color Differential Staining of Nor-Associated Heterochromatic Segments Using Acridine Orange

A new staining technique has been evaluated for detecting heterochromatic segments accompanying nucleolus organizing regions (NORs). This technique essentially consists of C-banding followed by acridine orange staining. When the technique was applied to five species of plants, the NOR-associated heterochromatic segments (NOR H-segments) were differentiated from other segments of the chromosomes as regions emitting yellowish green fluorescence. An incubation of at least 30 min in hot 2 × SSC was required to make the NOR H-segments emit yellowish green fluorescence in Nothoicordum fragrans. Fluorescence on other heterocnromatic segments varied from reddish orange to bright yellow; euchromatic segments emitted orange or yellowish orange fluorescence. The technique permits identification of NOR H-segments throughout mitosis based on the characteristic fluorescent color.     

Color differential staining of NOR-associated heterochromatic segments using acridine orange

A new staining technique has been evaluated for detecting heterochromatic segments accompanying nucleolus organizing regions (NORs). This technique essentially consists of C-banding followed by acridine orange staining. When the technique was applied to five species of plants, the NOR-associated heterochromatic segments (NOR H-segments) were differentiated from other segments of the chromosomes as regions emitting yellowish green fluorescence. An incubation of at least 30 min in hot 2 x SSC was required to make the NOR H-segments emit yellowish green fluorescence in Nothoscordum fragrans. Fluorescence on other heterochromatic segments varied from reddish orange to bright yellow; euchromatic segments emitted orange or yellowish orange fluorescence. The technique permits identification of NOR H-segments throughout mitosis based on the characteristic fluorescent color.     

A comparative study of quantitative stains for DNA in image cytometry.

In this study we examined the reproducibility of several stains used to measure nuclear DNA by image cytometry. The specimens were touch preparations of liver and testis from mouse and liver, intestine and brain from rat, fixed in either neutral formalin or Carnoy's solution. The tested stains included four Feulgen methods (pararosaniline, azure-A, thionin and acriflavine), the gallocyanine-chromalum stain and two fluorescent stains (acridine orange and propidium iodide). Absorbance measurements employed a video image analysis system; fluorescence measurements were from a scanning microspectrophotometer. The acriflavine-Feulgen stain was analyzed for both absorbance and fluorescence. All seven stains were quantitative for DNA and gave reproducible results. The absorbance measurements had a lower coefficient of variation (CV) than the fluorescence values. In a nested analysis of variance of the pararosaniline Feulgen stains, cell-to-cell variability accounted for 67% of the total variance; slide-to-slide, 9%; and batch-to-batch, 24%. These values did not change significantly when the staining was performed in an automatic staining machine. For DNA analysis using image cytometry, we conclude that the Feulgen staining technique is the most useful. In particular, acriflavine-Feulgen-stained cells fixed in Carnoy's fluid give the least variation between measurement values and the most accurate ratios between the separate ploidy groups. For fluorescence cytometry we recommend Carnoy's fixation and the acriflavine-Feulgen stain because of its narrow CV as compared to acridine orange and propidium iodide.     

Cytofluorometric and cytochemical comparisons of normal and abnormal human cells from the female genital tract.

Acridine orange staining of exfoliated cells from epithelial tissues facilitates discrimination between normal and abnormal cells: abnormal cells develop highly elevated nuclear fluorescence. Comparisons of acridine orange (AO) staining with propidium iodide (PI) or Feulgen staining have shown that: (a) PI staining also provides highly elevated nuclear fluorescence from abnormal cells; (b) the distributions of nuclear fluorescence following AO or PI staining were usually not significantly different as judged by the Kolmogorov-Smirnov test; (c) fluorescence emission spectra from AO and PI stained cells are consistent with the hypothesis that both fluorochromes bind to DNA within cell nuclei; (d) DNAse treatment of AO stained normal cells eliminates the nuclear fluorescence peak from slit-scan contours; RNAse treatment has no effect on nuclear fluorescence; (e) the distribution of abnormal cell nuclear fluorescence after AO staining is usually, but not always, significantly different from the distribution of abnormal cell nuclear absorbance after Feulgen staining, with relative nuclear fluorescence being greater than relative nuclear absorbance. The hypothesis currently most consistent with these results is that elevated Feulgen DNA content can account for only part of the discrimination provided by AO staining, and that the chromatin within abnormal cells is altered so as to increase accessibility of DNA to intercalating dyes.     

123 Permanent Fluorescent Nuclear Staining of Binucleate Dinoflagellates

Typically, fluorescent microscopy of dinoflagellate nuclei is of poor resolution, due mainly to visual obstruction of the nuclei by plastids, pigment granules, and thecal plates. Moreover, the usual slide mounts using buffered glycerol are temporary, and fade after a week or so. We have developed a procedure to clear pigments from dinoflagellates, followed by fluorescent staining of the nuclei. The cells are then prepared as permanent mounts using an ultraviolet light-catalyzed resin to produce stained samples which may be kept for at least three years with little loss of fluorescence. This procedure can also be used to prepare plastic embedded dinoflagellate cells which can then be sectioned at 1–2 nm, fluorescent stained, and permanently mounted. Suitable nuclear stains are DAPI, Hoechst 33258, ethidium bromide and acridine orange. The dinoflagellate (dinokaryotic), and endosymbiont (eukaryotic) nuclei are clearly visualized, revealing individual chromosomes in the dinoflagellate nucleus, and a highly lobed morphology of the endosymbiont nucleus.     

Hemocytes of the centipede Scutigera coleoptrata (Chilopoda,Notostigmophora) with notes on their interactions with the tracheae

The hemocytes of Scutigera coleoptrata were investigated by light and electron microscopy. Four types of hemocytes were identified: prohemocytes, plasmatocytes, granulocytes, and spherulocytes. Only granulocytes could be distinguished from the three other types by May-Grünwald staining, as this is the only hemocyte type demonstrating an eosinophilic reaction. Shape and size give further indications for distinguishing the cell types. In addition, differentiation is possible on the basis of their ultrastructure. However, only a combination of all three methods (staining and light and electron microscopy) allows clear separation of the cell types. As transitional stages between the cell types occur in S. coleoptrata, it is likely that prohemocytes, plasmatocytes, and granulocytes are ontogenetic stages of a single cell lineage. Special cell components and their possible functions are described. Plasmatocytes exocytose tubular structures that probably play a role in coagulation processes. These tubular structures develop in the grana of plasmatocytes. Also, a special arrangement of microtubules and microfilaments was demonstrated. For the first time interactions between hemocytes and tracheae are documented within the Chilopoda. It is assumed that the hemocytes meet their oxygen requirements directly from the tracheae. Phylogenetic implications of the results are discussed.     

Circulating Hemocytes from Larvae of the Japanese Rhinoceros Beetle Allomyrina dichotoma (Linnaeus) (Coleoptera: Scarabaeidae) and the Cellular Immune Response to Microorganisms

Hemocytes of the last larva of the Japanese rhinoceros beetle A. dichotoma (Linnaeus) (Coleoptera: Scarabaeidae) were classified as granulocytes, plasmatocytes, oenocytoids, spherulocytes, prohemocytes, and adipohemocytes. Among these cell types, only the granulocytes became immunologically activated with obvious morphological changes, displaying large amoeba-like, lobopodia-like, and fan-like structures. In addition, their cytoplasmic granules became larger and greatly increased in number. To explore whether these granules could be immunologically generated as phagosomes, total hemocytes were stained with LysoTracker. Greater than 90% of the granulocytes retained the LysoTracker dye at 4 h post-bacterial infection. In flow cytometry analysis, the red fluorescent signal was highly increased at 4 h post-bacterial infection (60.36%) compared to controls (5.08%), as was confirmed by fluorescent microscopy. After 12 h post-infection, these signals returned to basal levels. The uptake of pathogens by granulocytes rapidly triggered the translocation of the microtubule-associated protein 1 light chain 3 alpha (LC3) to the phagosome, which may result in enhanced pathogen killing.     

Fluorescence microscopy of viable mast cells stained with different concentrations of acridine orange.

Freshly harvested rat peritoneal mast cells were stained with different concentrations of acridine orange, a metachromatic fluorochrome known to form complexes with chromatin and muscopolysaccharides. Fluorescence metachromasia was observed in cytoplasmic granules in cell populations with intracelluar dye contents as low as 5 X 10(-16) mole per cell, one-half decade lower than required to produce metachromatic staining of the nucleus. Cytoplasmic granules did not stain uniformly throughout the cell; some granules exhibited red fluorescence and others green. As the amount of acridine orange uptake per cell was increased, cytoplasmic fluorescence became uniformly red and nuclear fluorescence gradually changed from green to yellow.     

Fluorescence microscopy of viable mast cells stained with different concentrations of acridine orange

Summary Freshly harvested rat peritoneal mast cells were stained with different concentrations of acridine orange, a metachromatic fluorochrome known to form complexes with chromatin and mucopolysaccharides. Fluorescence metachromasia was observed in cytoplasmic granules in cell populations with intracellular dye contents as low as 5×10^–16 mole per cell, one-half decade lower than required to produce metachromatic staining of the nucleus. Cytoplasmic granules did not stain uniformly throughout the cell; some granules exhibited red fluorescence and others green. As the amount of acridine orange uptake per cell was increased, cytoplasmic fluorescence became uniformly red and nuclear fluorescence gradually changed from green to yellow.     

Palmitate induces structural alterations in nuclei of cardiomyocytes

The objective of this study was to examine the hypothesis that the alterations of cardiac nuclei, that has been noted in some cardiomyopathies, can be produced by palmitate, a saturated fatty acid present in high circulating concentrations in patients with conditions associated with a high probability of developing cardiomyopathy. Cardiomyocytes isolated from embryonic chick ventricle were maintained in culture for 72 h and then treated with palmitate, 100 microM for 24 h. Cells were stained with acridine orange or Giemsa and examined microscopically. Cell size and nuclear size were examined by forward light scatter during flow cytometry. Cells were permeabilized and their nuclei were stained with propidium iodide and examined by flow cytometry on populations of 10,000 cells. Cardiomyocytes treated with palmitate displayed changes in nuclear appearance as nuclei were larger, relative to cell size, with more intense acridine orange staining in a peripheral location. Nucleoli were often disrupted. Palmitate produced a significant (P < 0.001) and 17% increase in nuclear size compared to untreated cells using flow cytometry analysing forward light scatter to estimate nuclear and whole cells size. There were no significant changes in the size of the whole cell and ratio of nucleus to whole cell was significantly (P < 0.01) increased compared to control cells. Fluorescent activating cell sorting analysis of propidium iodide stained nuclei demonstrated that the nuclear enlargement was not due to cell mitosis as the proportion of nuclei in Go/G1, S or M was not changed by palmitate. In summary, these studies identify that palmitate can induce structural abnormalities of cardiomyocytes nuclei by producing increased nuclear size and nucleolar destruction.