Variomacs magnet

The P. falciparum (ITO4/A4) laboratory clone was cultured in erythrocytes as previously described [16] (link), [17] (link). All cultures used in experiments were negative for Mycoplasma spp. based on the absence of mycoplasma enzyme activity using the MycoAlert Mycoplasma detection kit (Lonza Biologicals, Verviers, Belgium). Trophozoite and schizont stages of P. falciparum cultures were enriched to >90% purity by magnetic column isolation. Briefly, parasite cultures were centrifuged at 500 g for 5 minutes; the cell pellet was resuspended in 3 mL of supplemented serum-free RPMI-1640 (RPMI-SF containing 16 mM D-glucose (Sigma), 2 mM L-glutamine (Sigma), 37.5 mM HEPES (Lonza), 2.5 µg/mL gentamicin (Sigma), 1X HT supplement (Gibco, Invitrogen), pH 7.2–7.4) and applied to a LS column in a VarioMACS magnet (Milteny Biotec, Bergish-Gladbach, Germany). The column was washed with RPMI-SF until erythrocytes from the parasite culture were absent from the flow–through. The column was then removed from the magnet and the paramagnetic, parasitized cells were eluted in RPMI-SF. Purity of isolate was confirmed to be >90% by microscopic examination of Giemsa-stained thin film blood smear. All cultures used in experiments were negative for Mycoplasma spp.

The volume of saponin-isolated 3D7 trophozoites and of erythrocytes infected with 3D7 trophozoites was measured using a Beckman Coulter Multisizer 4 fitted with a 100 μm ‘aperture tube’. In the case of isolated parasites, the cells were washed and resuspended (at 37°C) in pH 7.10 Experimental Saline Solution. The electrolyte solution within the aperture tube was the same as the Experimental Saline Solution except that it lacked glucose. For experiments on infected erythrocytes, the infected cells were separated from uninfected erythrocytes using a Miltenyi Biotec VarioMACS Magnet [47 (link), 48 (link)] and the cells were maintained at 37°C in bicarbonate-free RPMI supplemented with 25 mM HEPES, additional glucose (11 mM) and 0.2 mM hypoxanthine, and pH-adjusted to 7.40. For these measurements the electrolyte solution within the aperture tube differed only from this medium in that it was not supplemented with hypoxanthine or additional glucose. For each measurement of cell volume, approximately 20,000 pulses (each corresponding to the passage of a single cell through the aperture) were recorded. The median volume of the cells within each sample was determined by fitting a log Gaussian distribution curve to the population data.

Upon thawing, cells were washed thrice in complete medium (RPMI-1640 containing 10% heat inactivated human serum, 2 nM L-glutamine, 100 U/ml of penicillin and 100 mg/ml of streptomycin sulPHAte), re-suspended in complete medium, counted using trypan blue (Sigma) and aliquoted into U-bottom 96-well plates.
For T cell proliferation assays, 2 × 10⁵ cells in 100 μl was added per well. Subsequently, 100 μl of purified fresh pRBCs at trophozoite/schizont stage (Pf 7G8, Pf NF54 or Plasmodium knowlesi A1H1.1) or uRBCs (6 × 10⁵ cells/well), 1% phytohaemagglutinin (PHA; Gibco) or media only was added, and PBMCs were cultured for 7 days at 37 °C, 5% CO₂. Each treatment was tested in triplicate.
For intracellular cytokine staining, 5 × 10⁵ cells in 100 μl were added per well. Subsequently, 100 μl of purified fresh Pf 7G8 pRBCs or uRBCs (1 × 10⁶ cells/well), 1% PHA or media only was added, and PBMCs were cultured for 36 h at 37 °C, 5% CO₂. Each treatment was tested in triplicate. Sorbitol-synchronised, Mycoplasma-negative, live, late-stage trophozoite/schizont stage pRBCs used in the above in vitro assays were purified by magnetic separation over CS columns (Miltenyi Biotec) on a VarioMACs magnet (Miltenyi Biotec) for these assays.