Separation of blood-cell-free trypanosomes and malaria parasites on a sucrose gradient

Williamson, Dr.J.; Cover, B.

doi:10.1016/0035-9203(66)90247-1

Cited by 34 publications

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“…The plasma was removed and the packed cells washed twice by resuspension and centrifugation in 4-5 volumes of cold Krebs glucose saline (KGS) (Krebs & Eggleston, 1940). The cells were then suspended in KGS at a 1 in 5 dilution and approximately 15 ml quantities of suspension were layered over 50 ml volumes of 0-7 M sucrose in KGS in 100 ml glass centrifuge tubes, before being centrifuged at 250 g for 7 min (modified from Williamson & Cover, 1966). Following centrifugation, the parasitized erythrocytes, which form a brown layer at the sucrose-saline interface, were removed with a Pasteur pipette and washed twice in KGS at 1500 g for 10 min.…”

Section: Proteins Synthesized By P Knowlesi In Vitro 179mentioning

confidence: 99%

“…The supernatant fluid, containing unincorporated [ 3 H]isoleucine, was removed by aspiration and the packed cells were washed twice by resuspension and centrifugation in 10 ml of ice-cold KGS. 'Free' parasites were produced by immune lysis and were generally separated from uninfected erythrocyte ghosts, leucocytes and membrane debris by centrifugation on 10 ml of 0-75 M sucrose (modified from the continuous 0-25-0-7 M sucrose gradient technique of Williamson & Cover, 1966). After 2 washes in ice-cold KGS, the 'free' parasites were suspended in approximately 0-3 ml of KGS and were either used immediately or were kept at -20 °C until required.…”

Section: Recovery Of Cells From Culturementioning

confidence: 99%

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Analysis of proteins synthesizedin vitroby the erythrocytic stages ofPlasmodium knowlesi

1980

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SUMMARYStudies were performed to identify specific parasite proteins synthesized withinPlasmodium knowlesi-infected rhesus erythrocytes. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of whole parasites freed from the host erythrocyte by immune lysis, of membranous and cytoplasmic parasite fractions, and of isolated merozoites, detected several parasite-specific components after Coomassie Blue staining of the separated proteins. However, significant contamination with host erythrocyte material generally occurred, particularly in the whole parasite and parasite membrane preparations. Improved identification of plasmodial proteins was subsequently afforded by a radioisotope labelling technique in which parasitized erythrocytes were cultivatedin vitrowith [3H] isoleucine prior to electrophoretic analysis. Of 11 principal labelled peaks ranging in molecular weight from approximately 17000 to 145000 which were detected upon electrophoresis of whole parasites harvested from culture, all were observed in the cytoplasmic fraction while at least 5 were also associated with the membranous cell fraction. Analysis of different developmental stages of the intra-erythrocytic parasite revealed no significant stage-specific qualitative variations in the electrophoretic profiles. Quantitatively, however, the middle to late trophozoites incorporated more [3H] isoleucine into protein than the other intra-erythrocytic stages. Analysis of merozoites purified from labelled schizonts showed a protein pattern similar to the other stages. This confirmed that host components did not contribute to the labelling pattern and that none of the labelled proteins were specific to the residual cytoplasm remaining after merozoite formation.

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Section: Proteins Synthesized By P Knowlesi In Vitro 179mentioning

confidence: 99%

Section: Recovery Of Cells From Culturementioning

confidence: 99%

Analysis of proteins synthesizedin vitroby the erythrocytic stages ofPlasmodium knowlesi

1980

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“…Buffy coat and plasma collected from patients at the PRCT were examined by the T. brucei bDNA assay. The use of buffy coat samples resulted in higher sensitivity than the use of plasma samples (data not shown), consistent with the cosedimentation of trypanosomes with leukocytes (44). Therefore, all buffy coat samples were examined in duplicate.…”

Section: Selection Of Targets and Optimization Of The Bdna Assaymentioning

confidence: 54%

“…To determine whether the bDNA assay detected bloodstream forms of T. brucei as effectively as it detected cultured procyclic forms, blood samples from infected rats were examined. We also analyzed various simple specimen preparations including whole blood, blood diluted with ammonium chloride to lyse (44). To facilitate comparison across sample types, the buffy coat was reconstituted by 10-fold dilution with PBS-glucose-BSA to approximate the initial volume of the sample; in routine processing, concentrated buffy coat would presumably be used.…”

Section: Selection Of Targets and Optimization Of The Bdna Assaymentioning

confidence: 99%

Detection of Trypanosoma brucei spp. in human blood by a nonradioactive branched DNA-based technique

et al. 1996

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We have developed a nonradioactive branched DNA (bDNA)-based assay for the diagnosis of the African trypanosomiases in simple buffy coat preparations of human blood. Two repetitive DNA sequences specific to the Trypanosoma brucei complex were chosen as targets of the bDNA assay, a technique which amplifies the signal from a target molecule rather than the target itself. Comparable sensitivities were observed with cloned target sequences, purified T. brucei DNA, procyclic trypanosomes, and bloodstream trypomastigotes. The results of bDNA analysis of human blood samples from Côte d'Ivoire (n ‫؍‬ 50) showed excellent agreement with those of buffy coat microscopy. The bDNA technology offers certain advantages over alternative molecular biological techniques, including the simplicity of sample preparation and of the procedure itself, the stability of the reagents, the ability to process large numbers of samples simultaneously, and freedom from crosscontamination artifacts. We have successfully applied the bDNA technique to the detection of T. brucei in clinical samples from regions where T. brucei infection is endemic; to our knowledge, this is the first report of the molecular detection of T. brucei in human blood.

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“…vivax, P. cynomolgi, P. knowlesi, P. berghei, P. chabaudi, P. vin-ckei, P. yoelii and P. gallinaceum. The gradients were com posed of sucrose (Williamson and Cover, 1966), bovine serum albumin (Rowley et al, 1967), Percoli (Knight and Sinden, 1982;Stanley et al, 1982;Rivadeneira et al, 1983), Ficoll (Eling, 1977;Mrema et al, 1979;Tosta et al, 1980) or metrizamide (Eugui and Allison, 1979).…”

Section: Introductionmentioning

confidence: 99%

Plasmodium berghei :the use of discontinuous urografin density gradients for the separation of exoerythrocytic malaria parasites

François

Desombere

Wéry

1993

Ann. Parasitol. Hum. Comp.

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Urografin was used in the lower cushion of discontinuous density gradient systems, for the separation of human hepatoma cells (Hep G2) infected with exoerythrocytic P. berghei forms from uninfected cells. The hepatoma cells exhibited a rather heterogeneous density distribution, masking the possible density differences between infected and uninfected cells and hindering the efficient separation of both cell types. Purely osmotic damage caused by Urografin on human erythrocytes and hepatoma cells is very limited. On the other hand, the direct toxic effects on P. falciparum blood stages and on P. berghei exoerythrocytic stages are very pronounced. The growth of the former forms is partially inhibited after a pretreatment, but remains acceptable if the contact with Urografin is relatively short. It is almost completely blocked during permanent incubation. The latter forms are killed after 1 hour of contact with Urografin.

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Separation of blood-cell-free trypanosomes and malaria parasites on a sucrose gradient

Cited by 34 publications

References 0 publications

Analysis of proteins synthesizedin vitroby the erythrocytic stages ofPlasmodium knowlesi

Analysis of proteins synthesizedin vitroby the erythrocytic stages ofPlasmodium knowlesi

Detection of Trypanosoma brucei spp. in human blood by a nonradioactive branched DNA-based technique

Plasmodium berghei :the use of discontinuous urografin density gradients for the separation of exoerythrocytic malaria parasites

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