Macs ld separation column

For IEM, mature schizonts from the DPAP3-GFP and 3D7 control lines were concentrated using a magnetic activated cell sorting (MACS) LD separation column (Miltenyi Biotec). Briefly, iRBCs were loaded onto an LD column attached to Midi MACS pre-equilibrated with media. The column was washed twice with media and schizonts eluted with media after detaching the column from the magnet. Parasites were then fixed in 4% paraformaldehyde/0.1% glutaraldehyde (Polysciences) in 100 mM PIPES and 0.5 mM MgCl₂, pH 7.2, for 1 h at 4°C. Samples were embedded in 10% gelatine and infiltrated overnight with 2.3 M sucrose/20% polyvinyl pyrrolidone in PIPES/MgCl₂ at 4°C. Samples were trimmed, frozen in liquid nitrogen, and sectioned with a Leica Ultracut UCT cryo-ultramicrotome (Leica Microsystems). Sections of 70 nm were blocked with 5% FBS and 5% NGS for 30 min and subsequently incubated with rabbit anti-GFP antibody 6556 (Abcam) at 1:750 overnight at 4°C. Colloidal gold conjugated anti rabbit (12 nm) IgG (Jack Imm Res Lab) was used as secondary antibody.

B cells were purified by negative selection, for which eight-week-old female mice were euthanized. Spleen cells were collected with cold RPMI (Gibco, Grand Island, NY, USA). The erythrocytes were eliminated using lysis buffer (Sigma−Aldrich), and the splenocytes were incubated with anti-CD43 (Ly-48) antibody conjugated with magnetic beads (Miltenyi Biotec). The cells went through a MACS LD separation column (Miltenyi Biotec). A >96% purity was determined through flow cytometry.