Plasmodium falciparum in Culture: Use of Outdated Erythrocytes and Description of the Candle Jar Method

Jensen, James B.; Trager, William

doi:10.2307/3279900

Cited by 302 publications

(147 citation statements)

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“…The Nigerian strain of Plasmodium falciparum [17] was maintained by serial passages in human erythrocytes at 7 % haematocrit according to the Petri-dish candle-jar method of Jensen and Trager [18] using RPMI 1640 medium supplemented with 25 mM Hepes buffer (pH 7n4) and 10 % human serum. Before each experiment, infected cells were maintained for at least 24 h in modified RPMI 1640 medium supplemented with 25 mM Hepes buffer (pH 7n4) (medium A) and 5 % human serum to maximally reduce the serine content.…”

Section: Biological Materialsmentioning

confidence: 99%

“…Radioactive precursors were incorporated by incubating P. falciparum-or P. knowlesi-infected suspensions (3-10 % and 9-100 % parasitaemia respectively) at 6-16 % final haematocrit in medium A (0n5 ml final volume) in microtubes according to the candle-jar method [18]. Radioactive precursors were usually present at 10 µM [$H]choline (44 Ci\mmol), 4-10 µM [$H]serine (0n5-2n5 Ci\mmol) or 2 µM [$H]Etn (2n5-5 Ci\mmol).…”

Section: Metabolic Studiesmentioning

confidence: 99%

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Phospholipid metabolism of serine in Plasmodium-infected erythrocytes involves phosphatidylserine and direct serine decarboxylation

Elabbadi

Ancelin

Vial

1997

Biochemical Journal

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Erythrocytes infected with Plasmodium falciparum or Plasmodium knowlesi efficiently incorporated radioactive serine into phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho). Serine was also metabolized into ethanolamine (Etn) and phosphorylethanolamine (P-Etn) via direct serine decarboxylation ; this is a major phenomenon since together these metabolites represent 60 % of total radioactive water-soluble metabolites. They were identified by reverse-phase HPLC and two TLC-type analyses and confirmed by alkaline phosphatase treatment, which depleted the radioactive P-Etn peak completely with a concomitant increase in that of Etn. In the presence of 5 µM labelled serine, radioactivity appeared in Etn and P-Etn after a 25 min lag period, and isotopic equilibrium was reached at 40 and 95 min respectively. There was a similar lag period for PtdEtn formation, which accumulated steadily for at least 180 min. Incorporation of serine into phospholipids and water-soluble metabolites increased in the presence of up to 500 µM external serine. An apparent plateau was then reached for all metabolites except intracellular serine and Etn. Exogenous Etn (at 20 µM) induced a concomitant dramatic decrease in serine incorporation into P-Etn and all phospholipids, but not

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Section: Biological Materialsmentioning

confidence: 99%

Section: Metabolic Studiesmentioning

confidence: 99%

Phospholipid metabolism of serine in Plasmodium-infected erythrocytes involves phosphatidylserine and direct serine decarboxylation

Elabbadi

Ancelin

Vial

1997

Biochemical Journal

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“…P. falciparum (FCR-3/Gambia) was cultured in 100mm petri dishes in a candle jar (7). Cultures of K+ and Kparasites were synchronized by treatment with sorbitol (8).…”

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confidence: 99%

Characterization of a protein correlated with the production of knob-like protrusions on membranes of erythrocytes infected with Plasmodium falciparum

Kilejian

1979

Proc. Natl. Acad. Sci. U.S.A.

201

105

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Membranes of erythrocytes infected with Plasmodium falciparum develop protrusions called "knobs." These protrusions are not apparent on erythrocytes infected with young parasites (rings) but develop with the growth of parasites to the trophozoite and schizont stages. The nature and origin of knobs were characterized by comparing the stagespecific proteins of two culture lines of P. falciparum, K+ and K-. K+ parasites produce knobs; K-parasites do not. Erythrocytes infected with both types of parasites were labeled metabolically and samples were analyzed by electrophoresis in sodium dodecyl sulfate/polyacrylamide gels. There were no apparent differences in Coomassie blue-stained or radioactive components of rings of K+ Membranes of erythrocytes infected with Plasmodium falciparum develop morphological alterations that were first described as "knob-like protrusions" (1). These protrusions are not apparent on erythrocytes that are infected with ring stages; they develop with the growth of parasites. In infected hosts, erythrocytes parasitized with multinucleate stages become sequestered and are not seen in the peripheral circulation. The knobs have been shown to be the sites of adhesion between sequestered erythrocytes and venous endothelial cells (2).A previous study (3) demonstrated that these protrusions were antigenically different from adjacent areas devoid of them. In an attempt to determine whether knob constituents were antigens of parasite origin that were incorporated into the host cell membrane, membrane fractions enriched in knobs were isolated from metabolically labeled infected erythrocytes and compared with fractions devoid of knobs. An additional labeled protein, with an apparent molecular weight of 70,000-80,000, was identified in samples enriched with knobs (4). From these results it could not be concluded with certainty whether this protein was a knob constituent or some other protein associated with membranes. Recently, it was noted that in cultures of an African strain of P. falciparum (FCR-3/Gambia) that had been maintained in flow vials (5) for >2 years by W. Trager some parasites failed to produce knobs (6). We were able to select for these parasites and obtain a culture line that showed mainly (about 99%) parasitized erythrocytes with smooth membranes (A. Kilejian and W. Trager, unpublished data). For purposes of discussion, the parent strain will be referred to as K+ and the derived culture line that does not produce knobs, as K-.This communication is a report on the comparison of stagespecific proteins of K+ and K-parasites. The results show that a metabolically labeled protein, of -80,000 molecular weight, that had been noted in a previous study (4) is correlated with the production of knobs. Two additional components that are synthesized during the development of merozoites have been identified.MATERIALS AND METHODS Parasites. P. falciparum (FCR-3/Gambia) was cultured in 100mm petri dishes in a candle jar (7). Cultures of K+ and Kparasites were synchronized by treatment with s...

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“…

AbstractUnlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1 . While the parasitemia achieved can reach the ≈40% limit, the investigator usually keeps the percentage at around 10%.

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confidence: 99%

Separation of <em>Plasmodium falciparum</em> Late Stage-infected Erythrocytes by Magnetic Means

Coronado

Tayler

Corrêa

et al. 2013

JoVE

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Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1 . While the parasitemia achieved can reach the ≈40% limit, the investigator usually keeps the percentage at around 10%. In many cases it is necessary to isolate the parasite-containing red blood cells (RBCs) from the uninfected ones, to enrich the culture and proceed with a given experiment.When P. falciparum infects the erythrocyte, the parasite degrades and feeds from haemoglobin 2,3 . However, the parasite must deal with a very toxic iron-containing haem moiety 4,5 . The parasite eludes its toxicity by transforming the haem into an inert crystal polymer called haemozoin 6,7 . This iron-containing molecule is stored in its food vacuole and the metal in it has an oxidative state which differs from the one in haem 8 . The ferric state of iron in the haemozoin confers on it a paramagnetic property absent in uninfected erythrocytes. As the invading parasite reaches maturity, the content of haemozoin also increases 9 , which bestows even more paramagnetism on the latest stages of P. falciparum inside the erythrocyte.Based on this paramagnetic property, the latest stages of P. falciparum infected-red blood cells can be separated by passing the culture through a column containing magnetic beads. These beads become magnetic when the columns containing them are placed on a magnet holder. Infected RBCs, due to their paramagnetism, will then be trapped inside the column, while the flow-through will contain, for the most part, uninfected erythrocytes and those containing early stages of the parasite.Here, we describe the methodology to enrich the population of late stage parasites with magnetic columns, which maintains good parasite viability 10 . After performing this procedure, the unattached culture can be returned to an incubator to allow the remaining parasites to continue growing. Video LinkThe video component of this article can be found at http://www.jove.com/video/50342/ ProtocolAll steps of the protocol, except for centrifugations, should be carried out inside a hood to keep the sample sterile. Late Stage Isolation of P. falciparum-infected ErythrocytesAll late stages of Plasmodium-infected erythrocytes can be separated with this methodology, since hemozoin, which confers paramagnetism on the parasite, is a common metabolite to the genus. A high parasitemia (3-10%) in culture is recommended to get better yields with this protocol, although a typical culture will contain 2-4% hematocrit (volume percentage of red blood cells) and 1-8% parasitemia (percentage of infected red blood cells). Parasites can be cultured as described by Trager and Jansen 1 .1. A simple, multi-part setup must be assembled for every schizont isolation procedure. For this, LS columns, a Magnetic MidiMACS Separator and a MACS MultiStand from Miltenyi BioTec (Figure 1) are used. First, attach the magnetic separator to the metal stand and then place the magnetic column on the separator. 2. In a separate tube (use 50 ml conicals throu...

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Plasmodium falciparum in Culture: Use of Outdated Erythrocytes and Description of the Candle Jar Method

Cited by 302 publications

References 3 publications

Phospholipid metabolism of serine in Plasmodium-infected erythrocytes involves phosphatidylserine and direct serine decarboxylation

Phospholipid metabolism of serine in Plasmodium-infected erythrocytes involves phosphatidylserine and direct serine decarboxylation

Characterization of a protein correlated with the production of knob-like protrusions on membranes of erythrocytes infected with Plasmodium falciparum

Separation of <em>Plasmodium falciparum</em> Late Stage-infected Erythrocytes by Magnetic Means

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