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A quick, simple method for purifying Leishmania mexicana amastigotes in large numbers

Published online by Cambridge University Press:  06 April 2009

D. T. Hart ,
K. Vickerman  and
G. H. Coombs
D. T. Hart
Affiliation:
Department of Zoology, University of Glasgow, Glasgow G12 8QQ
K. Vickerman
Affiliation:
Department of Zoology, University of Glasgow, Glasgow G12 8QQ
G. H. Coombs
Affiliation:
Department of Zoology, University of Glasgow, Glasgow G12 8QQ
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Summary

A rapid method for the bulk isolation of purified Leishmania mexicana mexicana amastigotes from parasite-induced lesions in experimentally infected mice is described. The procedure includes purification steps based on differences in net cell charge, lysis susceptibility and buoyant density between parasite and host cells. Yields of up to 2 × 1010 untransformed amastigotes with minimal contamination with host cells and cell debris can be obtained. At least 90 % of the purified amastigotes are viable as judged by light and electron microscopy, the staining of their lysosomes with acridine orange, their ability to transform to promastigotes and their infectivity to macrophages in vivo and in vitro.

Type
Research Article
Information
Parasitology , Volume 82 , Issue 3 , June 1981 , pp. 345 - 355
Copyright
Copyright © Cambridge University Press 1981

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References

REFERENCES

Alexander, J. & Vickerman, K. (1975). Fusion of host cell secondary lysosomes with the parasitophorous vacuoles of Leishmania mexicana-infected macrophages. Journal of Protozoology 22, 502–8.CrossRefGoogle ScholarPubMed
Allison, A. C. & Young, M. R. (1969). Vital staining and fluorescence microscopy of lysosomes. In Lysosomes in Biology and Pathology, vol. 2 (ed. Dingle, J. T. and Fell, H. B.), pp. 600–28. Amsterdam: North Holland Publishing Company.Google Scholar
Berens, R. L., Brun, R. & Krassner, S. M. (1976). A simple monophasic medium for axenic culture of hemoflagellates. Journal of Parasitology 62, 360–5.CrossRefGoogle ScholarPubMed
Brazil, R. P. (1978). Isolation of the intracellular stages of Leishmania mexicana amazonensis using cellulose column. Annals of Tropical Medicine and Parasitology 72, 1–2.CrossRefGoogle ScholarPubMed
Childs, G. E., McRoberts, M. J. & Foster, K. A. (1976). Partial purification of amastigotes from cutaneous lesions of American leishmaniasis. Journal of Parasitoslogy 62, 676–9.CrossRefGoogle ScholarPubMed
Coombs, G. H. (1980). Endopeptidases of Leishmania mexicana mexicana amastigotes and promastigotes. Parasitology 81, lix.Google Scholar
Fulton, J. D. & Joyner, L. P. (1949). Studies on protozoa. Part 1. The metabolism of Leishman-Donovan bodies and flagellates of Leishmania donovani. Transactions of the Royal Society of Tropical Medicine and Hygiene 43, 273–86.CrossRefGoogle Scholar
Gero, A. M. & Coombs, G. H. (1980). Orotate phosphoribosyltransferase and orotidine-5'- phosphate decarboxylase in two parasitic kinetoplastid flagellates. Febs Letters 118, 130–2.CrossRefGoogle ScholarPubMed
Ghafoor, M. Y. & Coombs, G. H. (1980). Superoxide dismutase, peroxidase and glutathione metabolism in Leishmania mexicana mexicana amastigotes and promastigotes. Parasitology 81, xxviii.Google Scholar
Hart, D. T. & Coombs, G. H.(1980 a). Energy metabolism of Leishmania mexicana mexicana amastigotes and promastigotes. Parasitology 81, iv.Google Scholar
Hart, D. T. & Coombs, G. H. (1980 b). Morphological and biochemical studies of the in vitro transformation of Leishmania mexicana mexicana amastigotes to promastigotes. Journal of Protozoology, (in the Press).Google Scholar
Hart, D. T., Ghafoor, M. Y., Craft, J. A. & Coombs, G. H. (1980). Subcellular fractionation of Leishmania mexicana mexicana. Parasitology 81, lx.Google Scholar
Homewood, C. A. & Neame, K. D. (1976). A comparison of methods used for the removal of white cells from malaria-infected blood. Annals of Tropical Medicine and Parasitology 70, 249–51.CrossRefGoogle ScholarPubMed
Howard, R. J., Smith, P. M. & Mitchell, G. F. (1978). Removal of leukocytes from red cells in Plasmodium berghei-infected mouse blood and purification of schizont-infected cells. Annals of Tropical Medicine and Parasitology 72, 573–5.CrossRefGoogle ScholarPubMed
Infante, R. B., Hernandez, A. G., Riggione, F. & Dawidowicz, K. (1980). A new method for the partial purification of leishmania amastigotes from cutaneous lesions. Parasitology 80, 105–12.CrossRefGoogle ScholarPubMed
Krassner, S. M. (1966). Cytochromes, lactic dehydrogenase and transformation in Leishmania. Journal of Protozoology 13, 286–90.CrossRefGoogle ScholarPubMed
Murray, H. W. & Cohn, Z. A. (1979). Macrophage oxygen-dependant antimicrobial activity 1. Susceptibility of Toxoplasma gondii to oxygen intermediates. Journal of Experimental Medicine 150, 938–49.CrossRefGoogle Scholar
Robinson, J. & Cooper, J. M. (1970). Method of determining oxygen concentrations in biological media, suitable for calibration of the oxygen electrode. Analytical Biochemistry 33, 390–9.CrossRefGoogle ScholarPubMed
Simpson, L. (1968). The leishmania-leptomonad transformation of Leishmania donovani; nutritional requirements, respiratory changes and antigenic changes. Journal of Protozoology 15, 201–7.CrossRefGoogle Scholar
von Brand, T. (1979). Biochemistry and Physiology of Endoparasites. Amsterdam: Elsevier/North Holland Biomedical Press.Google Scholar
Walter, R. D., Buse, E. & Ebert, F. (1978). Effect of cyclic AMP on transformation and proliferation of Leishmania cells. Tropenmedizin und Parasitologie 29, 439–42.Google ScholarPubMed