Macs magnetic separation column

Merozoites were purified as previously described (31 (link)). Briefly, highly synchronized ring-stage PfPKAc:loxP parasites were treated with DMSO or rapamycin and grown until they became late-stage schizonts. Mature schizonts were isolated from uninfected erythrocytes using a MACS magnetic separation column (Miltenyi Biotec, Gladbach, Germay). Schizonts were treated with E64 cysteine protease inhibitor (10 μM) for 4–6 h to prevent egress. Merozoites were purified from schizont preparations by passage through a 1.2-μm-pore-size syringe filter (Acrodisc) (32 mm, Pall). Filtrate containing purified merozoites was added to uninfected erythrocytes (final concentration, 1% hematocrit), and suspensions were agitated at 37°C for 20 min. Suspensions were then plated out in triplicate (100 μl per well, topped up with media to total 200 μl). After 24 to 40 h, levels of parasitemia were determined by flow cytometry as described above. The concentrations of merozoites and erythrocytes were quantitated using CountBright Absolute counting beads (Life Technologies), and the invasion rate was calculated as follows: percentage of erythrocytes invaded × [(number of erythrocytes per microliter)/(number of merozoites per microliter)] (31 (link)).

Cultures (30 ml) of highly synchronous 3D7 WT or NF54 PKAloxP ring-stage parasites were prepared and treated with either DMSO (Sigma-Aldrich, MO) or rapamycin (LC Laboratories, MA). Once parasites had developed to become late schizonts, parasites were isolated from uninfected erythrocytes using a MACS magnetic separation column (Miltenyi Biotec, Gladbach, Germay). Cultures were centrifuged at 500 relative centrifugal force (RCF) for 5 min, and the parasite pellet was diluted 1:1,000 in fresh RPMI-HEPES. A 200-μl volume of RPMI-HEPES (0.25% hematocrit) was placed into microscope viewing chambers (Ibidi). A 50-μl volume of parasite suspension was applied to the fresh erythrocytes just prior to imaging. Parasites were imaged on an inverted Zeiss Live Cell AxioObserver microscope under bright-field conditions. The sample chamber was heated to 37°C and supplied with a humidified atmosphere that included 1% O₂, 5% CO₂, and 94% N₂. Quantitation of live-imaging experiments was performed using Image J software.

The isolation of silver nanoparticles from Magnetospirillum magnetotacticum culture was performed using MACS magnetic separation column (Miltenyl Biotec, Magdeburg, Germany). Briefly, M. magnetotacticum bacterial cells were suspended in 20 mM HEPES-4 mM EDTA (pH 7.4) and broken open by sonication for 5 min at 21 °C. The unbroken cells were removed by centrifugation at 9000 rpm for 30 min. The supernatant was harvested and passed through the MACS magnetic separation column, following the manufacturer’s protocol. Unbound magnetic particles were washed using 10 mM HEPES-200 mM NaCl (pH 7.4), and thereafter the silver nanoparticles were eluted with 10 mM HEPES (pH 7.4).

Peripheral blood mononuclear cells (PBMC) were isolated using methods previously described (Yarilina et al, 2011 (link)). Briefly, 60 ml of blood was collected from independent healthy human donors into heparinized tubes. The human blood was diluted 1:1 with PBS, and leukocytes were isolated using UNI-SEP maxi tubes (NOVAmed) following manufacturer's instructions. Briefly, UNI-SEP maxi tubes were spun at 1,000 g for 20 min and the mononuclear cell layer was retrieved using a pipette. The isolated cells were then washed with RPMI 1640, and selection for CD14⁺ cells was performed using the MACS magnetic separation columns (Miltenyi Biotec) as per manufacturer's instructions. Subsequently, the CD14⁺ cells were seeded in 96-well tissue culture plates at 1 × 10⁵ cells/well in 100 μl RPMI 160 supplemented with 10% FBS containing 100 IU/ml penicillin, 100 μg/ml streptomycin, and 20 ng/ml macrophage colony-stimulating factor (M-CSF)-1 (Life Technologies). The tissue culture media was replaced on day 5 and on day 7, and PBMC-derived macrophages were directly infected with GBS strains or primed with LPS (100 ng/ml) for 3 h before exposure to purified pigment (see below).