In vitro Culture of P. falciparum NF54 Parasites

P. falciparum NF54 parasites (27 (link)) were grown in vitro in O Rh⁺ erythrocytes at 37°C in a controlled atmosphere, using complete culture medium (RPMI 1640 supplemented with 0.5% AlbuMax II [Life Technologies BV, Nærum, Denmark]), essentially as described previously (28 (link)). The P. falciparum NF54-derived and pVBH-transfected clone G6 was generated as described elsewhere (29 (link)– (link)31 (link)). It was maintained in the same way as the parental strain P. falciparum NF54, except that blasticidin (10 mg/ml; Life Technologies) was added to shut down transcription of endogenous var genes and to erase the epigenetic memory. IEs were selected for surface expression of defined PfEMP1 proteins by immunomagnetic selection using (i) PfEMP1-specific rat antisera followed by biotinylated anti-rat antibody (Dako) and streptavidin-coupled Dynabeads (Fisher Scientific) or (ii) PAM1.4 followed by protein A-coupled Dynabeads (Fisher Scientific), essentially as described previously (32 (link)). For selection of IgM-binding IEs, we used human IgM (Sigma) coupled to M-450 epoxy beads (Life Technologies) according to the manufacturers' instructions. The genotypic identity of the parasites and the absence of Mycoplasma contamination were verified regularly as described previously (33 (link)).

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Jeppesen A., Ditlev S.B., Soroka V., Stevenson L., Turner L., Dzikowski R., Hviid L, & Barfod L. (2015). Multiple Plasmodium falciparum Erythrocyte Membrane Protein 1 Variants per Genome Can Bind IgM via Its Fc Fragment Fcμ. Infection and Immunity, 83(10), 3972-3981.

Publication 2015

blasticidin Dynabeads human IgM

Anti antibody Antisera Atmosphere Clone Culture medium Epigenetic memory Epoxy Erythrocytes Genes transcription Genotypic Human Mycoplasma Parasites Parental Protein a Strain Streptavidin Surface proteins

Corresponding Organization :

Other organizations : Copenhagen University Hospital, University of Copenhagen, Rigshospitalet

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Variable analysis

independent variables

Use of P. falciparum NF54 parasites
Growth of parasites in vitro in O Rh⁺ erythrocytes at 37°C in a controlled atmosphere
Use of complete culture medium (RPMI 1640 supplemented with 0.5% AlbuMax II)
Use of P. falciparum NF54-derived and pVBH-transfected clone G6
Addition of blasticidin (10 mg/ml) to shut down transcription of endogenous var genes and erase the epigenetic memory
Selection of IEs for surface expression of defined PfEMP1 proteins using PfEMP1-specific rat antisera or PAM1.4
Selection of IgM-binding IEs using human IgM coupled to M-450 epoxy beads

dependent variables

Growth and maintenance of P. falciparum parasites
Surface expression of defined PfEMP1 proteins on IEs
IgM-binding properties of IEs

control variables

O Rh⁺ erythrocytes
Controlled atmosphere at 37°C
Genotypic identity of the parasites and absence of Mycoplasma contamination

controls

Parental strain P. falciparum NF54 as a control for the NF54-derived and pVBH-transfected clone G6

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