The rapid isolation from human blood of concentrated, white-cell-free preparations of Plasmodium falciparum

Williamson, J.; Cover, B.

doi:10.1016/0035-9203(75)90014-0

Cited by 11 publications

(3 citation statements)

References 26 publications

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“…Fractionation techniques (Kass etal. 1971;Williamson & Cover, 1975;Hommel etal. 1979;Knight & Sinden, 1982;Vermeulen, Ponnudurai, Lensen, Roeffen & Meuwissen, 1983) have proved to be of variable efficiency and none produce material absolutely free of asexual stages such that the metabolism of the gametocyte may be studied with confidence.…”

Section: Discussionmentioning

confidence: 99%

“…Separation techniques have used either column chromatography (Kass, Wilierson, Riekmann, Carson & Becker, 1971) or differential centrifugation methods (Williamson & Cover, 1975;Hommel, Selkirk & Miltgen, 1979;Knight & Sinden, 1982). Despite the success of these methods, which have produced pure preparations of near mature stage IV and mature stage V gametocytes (Hawking et al 1971) that retain as much as 76% of their ability to exflagellate (viability), three practical difficulties are encountered.…”

Section: Introductionmentioning

confidence: 99%

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Gametocytogenesis of Plasmodium falciparum in vitro: a simple technique for the routine culture of pure capacitated gametocytes en masse

Ponnudurai

et al. 1984

Parasitology

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SUMMARYA technique is described for the growth of pure gametocyte cultures of Plasmodium falciparum. Using the classification of Hawking, Wilson & Gammage (1971) these cultures contain gametocytes of stages III, IV and V alone. Routine sexual cultures, varying from 0·35 ml static cultures to 500 ml shaking cultures, are exposed to an inhibitor of DNA synthesis (mitomycin C at 10 μg/ml) on the 11th day of culture, and the culture is harvested on the 14th day when capacitated stage V gametocytes are present. All other stages are killed by the drug and are morphologically degenerate within 2 days of the addition of the inhibitor.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Gametocytogenesis of Plasmodium falciparum in vitro: a simple technique for the routine culture of pure capacitated gametocytes en masse

Ponnudurai

et al. 1984

Parasitology

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“…1,2 The major limitation of these techniques is their inability to maintain physiologic osmolality throughout the purification procedure and avoid cell damage. 3 Percoll, a colloidal solution of polyvinylpyrrolidone-coated silica particles, can be used to overcome this problem of osmotically induced cell damage because of its low osmolality, 10 mOsm at 1.13 g/ml.…”

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confidence: 99%

Plasmodium falciparum: purification of the various gametocyte developmental stages from in vitro-cultivated parasites.

Kariuki

Kiaira.²,

Mulaa³

et al. 1998

The American Journal of Tropical Medicine and Hygiene

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Abstract. Cultivated Plasmodium falciparum gametocytes reach maturity in vitro in approximately 14-16 days, during which they pass through five morphologically distinct developmental stages. Purification of the earlier developmental stages has not been previously reported. We have modified the standard discontinuous Percoll gradient method for the separation of stage IV and V gametocytes to obtain enriched preparations of those and the earlier P. falciparum gametocyte stages. In contrast to the stages II, III, and IV, the mature stage V gametocytes from our gradient readily transformed into gametes. Such preparations may be useful in research studies on the mechanisms that underlie gametocytogenesis.Several previously reported techniques for the purification of mature Plasmodium falciparum gametocytes exploit the differences in sedimentation rates and buoyant densities of uninfected erythrocytes and erythrocytes infected with the various stages of the parasite. 1,2 The major limitation of these techniques is their inability to maintain physiologic osmolality throughout the purification procedure and avoid cell damage. 3 Percoll, a colloidal solution of polyvinylpyrrolidone-coated silica particles, can be used to overcome this problem of osmotically induced cell damage because of its low osmolality, 10 mOsm at 1.13 g/ml. 4 A Percoll step gradient centrifugation method has been successfully used to obtain highly purified preparations of viable mature P. falciparum and P. yoelii gametocytes. 5 The addition of fresh erythrocytes to P. falciparum cultures leads to loss of synchrony. 6,7 Synchrony can be restored by the addition of DNA inhibitors during gametocytogenesis. 8 However, inhibitors such as mitomycin C and chloroquine are toxic to stages I and II gametocytes. Mature gametocytes that have been synchronized by the addition of mitomycin C cannot be used immediately for research studies and membrane feeds because gametogenesis is inhibited for 24-48 hr after removal of the drug. 8 To avoid this transient drug toxicity, Ponnudurai and others synchronized gametocytes in an automated culture system by using gelatin flotation and N-acetyl glucosamine treatment. 9 Despite the fact that methods for synchronization and purification of the mature sexual stages of P. falciparum cultures have been separately described, these two techniques have not been combined to obtain purified fractions of the early gametocyte stages. This is a crucial requirement for research because the events that regulate gametocytogenesis are still in question. Our ability to study the early sexual stages, soon after morphologic differentiation, may provide insight into this intriguing aspect of cell cycle regulation. We have modified the Percoll step gradient technique to obtain enriched fractions of almost all the stages of P. falciparum gametocytes from in vitro culture. MATERIALS AND METHODS Culture of gametocytes.Plasmodium falciparum strain NF54 was cultured in vitro according to the Ifediba and Vandeberg 10 modification of the Trager and ...

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Plasmodium yoelii and Plasmodium berghei: Isolation of infected erythrocytes from blood by colloidal silica gradient centrifugation

Tosta

Sedegah

Henderson

et al. 1980

Experimental Parasitology

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The rapid isolation from human blood of concentrated, white-cell-free preparations of Plasmodium falciparum

Cited by 11 publications

References 26 publications

Gametocytogenesis of Plasmodium falciparum in vitro: a simple technique for the routine culture of pure capacitated gametocytes en masse

Gametocytogenesis of Plasmodium falciparum in vitro: a simple technique for the routine culture of pure capacitated gametocytes en masse

Plasmodium falciparum: purification of the various gametocyte developmental stages from in vitro-cultivated parasites.

Plasmodium yoelii and Plasmodium berghei: Isolation of infected erythrocytes from blood by colloidal silica gradient centrifugation

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