Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

Summary

The paramagnetic properties of hemozoin are used to isolate late stages of Plasmodium falciparum-infected red blood cells growing in culture. The method is simple and fast and does not affect the subsequent invasive capabilities of the parasites.

Abstract

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.

Protocol

All steps of the protocol, except for centrifugations, should be carried out inside a hood to keep the sample sterile.

1. Late Stage Isolation of P. falciparum-infected Erythrocytes

All 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 blo....

Results

In Figure 2, the culture passing through the magnetic column is shown, before (A) and after the procedure (B). One to two infected erythrocytes are usually seen on a 100X magnification field as shown in Figure 2, with the arrows pointing to infected erythrocytes in Figure 2A. In a typical procedure, starting with a culture at 5% parasitemia (Figure 2A), the performance of this procedure usually produces erythrocytes with.......

Discussion

In vitro cultures of the malaria parasite P. falciparum exhibit a limited parasitemia, with more than half of the red blood cells uninfected at the highest proliferation point of culture. For most research experiments, it is desirable to work only with the infected erythrocytes. To this end, a separation technique is necessary to divide the culture according to infection. Useful methods include the use of streptolysin O to permeabilize and lyze the uninfected RBCs ¹¹ and a series of variation.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RPMI 1640 Hepes Modified	Sigma-Aldrich	R4130	Supplemented with 10% human serum, 2% glucose, and 0.2% sodium bicarbonate
MidiMACS Separator	MACS Miltenyi BioTec	130-042-302
MACS MultiStand	MACS Miltenyi BioTec	130-042-303
LS Columns	MACS Miltenyi BioTec	130-042-401
Hemacytometer	Grafco	Grafco Neubauer Chamber	Can be found through many other suppliers