Summary
Cell surface glycosaminoglycans (GAGs) were measured, after various treatments, by their binding to Acridine Organge using flow cytometry. Using a critical electrolyte concentration and combining it with specific degradation of individual GAG elements, it was found possible to differentiate between GAG components. The technique was adapted for electron microscopy level to reveal characteristics of membrane-associated GAG. By this means, the cell membrane of the human leukaemic cell line K562 was shown to contain a large amount of GAG; 75% of it was highly sulphated GAG, mostly heparan sulphate. This component was evenly distributed in the outer plasma membrane layer. In the presence of other GAGs, the appearance of complex proteoglycan granules was detected.
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Culp, L., Rollins, B., Baniel, J. &Hitri, S. (1978) Two functionally distinct pools of glycosaminoglycans in the substrate adhesion site of murine cells.J. Cell Biol. 79, 788–801.
Giordana, M., Bertolotto, A., Mauro, A., Migheli, A., Pezzota, S., Racagni, G. &Schiffer, D. (1982) Glycosaminoglycans in human cerebral tumours.II. Histochemical findings and correlations.Acta Neuropath. (Berl.) 57, 299–305.
Hurst, R. E., Parmley, R. T., Nakamura, N., West, S. S., &Denys, F. R. (1981) Heparan sulfate of AH-130 ascites hepatoma cells: a cell-surface glycosaminoglycan not displaced by heparin.J. Histochem. Cytochem. 29, 731–7.
Kraemer, P. M. &Tobey, R. A. (1972) Cell-cycle dependent desquamation of heparan sulfate from the cell surface.J. Cell Biol. 55, 713–7.
Lüllmann-rauch, R. (1982) Histochemical evidence for lysosomal storage, of acid glycosaminoglycans in splenic cells of rats treated with Tilorone.Histochemistry 76, 71–87.
Metezau, PH., Djavadi-Ohaniance, L. &Goldberg, M. E. (1982) The kinetics and homogenicity of endocytosis of a receptor-bound ligand in a heterogeneous cell population studied by flowcytometry.J. Histochem. Cytochem. 30, 350–63.
Nakamura., N., Hurst, R. E., Corliss, D. A., Golden, J. F., Menter, J. M., & West, S. S. (1979) Chemical and cytochemical studies of heparan sulfates from AH-130 ascites hepatoma. InGlycoconjugate Research, p. 911. New York.
Okaya, T. &Kaneko, Y. (1970) Novel hyaluronidase fromStreptomyces.Biochim. biophys. Acta 198, 107–9.
Saito, H., Yamagata, T. &Suzuki, S. (1968) Enzymatic methods for the determination of small quantities of isomeric chrondroitin sulfatesJ. biol. Chem. 243, 2536–42.
Sannes, P. L., Katsuyama, T. &Spicer, S. S. (1978) Tannic acid-metal salt sequences for light and electron microscopic localisation of complex carbohydrates.J. Histochem. Cytochem. 26, 55–61.
Saunders, A. M. (1964) Histochemical identification of acid mucopolysaccharides with acridine orange.J. Histochem. Cytochem. 12, 164–70.
Scott, J. E. &Dorling, J. (1965) Differential staining of acid glycosaminoglycans (mucopolysaccharides) by alcian blue in salt solutions.Histochemie 5, 221–33.
Scott., J. E. Quintarelli, G. &Dellovo, M. C. (1964) The chemical and histochemical properties of Alcian blue. I. The mechanism of Alcian blue staining.Histochemie 4, 73–85.
Shepard, N. &Mitchell, N. (1976a) Simultaneous localization of proteoglycan by light and electronmicroscopy using toluidine blue O. A study of epiphyseal cartilage.J. Histochem. Cytochem. 24, 621–9.
Shepard, N. &Mitchell, N. (1976b) The localization of proteoglycan by light and electronmicroscopy using safranin O.J. Ultrastruct. Res. 54, 451–60.
Shepard, N. &Mitchell, N. (1981) Acridine orange stabilisation of glycosaminoglycans in beginning endochondrial ossification.Histochemistry 70, 107–14.
Shively, J. E. &Conrad, H. E. (1976) Formation of anhydrosugars in the depolymerisation of heparin.Biochem. 15, 3932–42.
Simionescu, M., Simionescu, N., Silbert, J. E. &Palade., G. E. (1981) Differentiated microdomains on the luminal surface of the capillary endothelium. II. Partial characterisation of their anionic sites.J. Cell Biol. 90, 614–21.
Spicer, S. S., Hardin, J. H. &Setser, M. E. (1978) Ultrastructural visualization of sulphated complex carbohydrates in blood and epithelial cell with high-iron diamine procedure.Histochem. J. 10, 435–52.
Spicer, S. S., Baron, D. A., Sato, A. &Schulte, B. A. (1981) Variability of cell surface glyconjugates. Relation to differences in cell function.J. Histochem. Cytochem. 29, 994–1002.
Takagaki, M., Parmley, R. T. &Denys, F. R. (1983) Ultrastructural cytochemistry and immunochemistry of proteoglycans associated with epiphyseal cartilage calcification.J. Histochem. Cytochem. 31, 1089–100.
Timar, J., Gyapay, G. &Lapis, K. (1979) Acridine orange staining of the mammalian fibroblast cell coat.Histochemistry 64, 189–93.
Timar, J., Lapis, K. & Csikos, A. (1980) SEM studies on acridine orange staining of the cell coat. InElectron Microscopy, Vol. 2 (edited byBroderoo, P. andPavert, W.), pp. 324–5. Leiden:
Timar, J., Podmaniczky, E., Lapis, K., Csikos, A. &Köszegvary, E. (1982) Cytochemical differences on RL 19 rat liver cells of different stage of growth. InElectron Microscopy, Vol. 3, pp. 356–7. Hamburg: Congress Organization Committee.
Toole, B. P., Biwes, C. &Gross, J. (1979) Hyaluronate and invasiveness of the rabbit V2 carcinoma.Proc. Natn Acad. Sci. USA 76, 6299–303.
Toole, B. P. (1981) Glycosaminoglycans in morphogenesis. InCell Biology of Extracellular Matrix (edited byHay, E. D.), pp. 259–94. New York, London: Plenum Press.
Underhill, C. B. &Keller, J. M. (1976) Density-dependent changes in the amount of sulfated glucosaminoglycans associated with mouse 3T3 cells.J. Cell Physiol. 89, 53–64.
West, S. S., Goldon, J. F., Menter, J. M. &Love, L. D. (1976) Quantitation of fluorescence fading phenomena for identifying intracellular biopolymers.J. Histochem. Cytochem. 24, 59–63.
West, S. S., Hurst, R. E. &Menter, J. M. (1977) Thermodynamics of mucopolysaccharides-dye binding. I. Dermatan sulphate-acridine orange system: direct measurement of free and bound dye.Biopolymers 16, 685–93.
Yanagisita, M. &Hascall, V. C. (1983) Characterisation of heparan sulfate proteoglycans synthesized by rat ovarian granulosa cell in culture.J. biol. Chem. 258, 12857–64.
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Timar, J., Boldog, F., Kopper, L. et al. Flow cytometric measurements and electron microscopy of cell surface glycosaminoglycans using Acridine Orange. Histochem J 17, 71–79 (1985). https://doi.org/10.1007/BF01003404
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DOI: https://doi.org/10.1007/BF01003404