P. falciparum was cultured as described previously16 (link),17 (link). Cultures were grown in RPMI-1640 Supplemented with 24 mM sodium bicarbonate (Sigma-Aldrich, St Louis, MO, USA) and 10% human serum (Interstate Blood Bank Inc, Memphis, TN, USA). They were gassed with 5% CO2/1% O2 and 94% N2 mixture and maintained at 37 °C. The ICAM-1/CD36 binding parasite strain ItG was used throughout18 (link).
Parasites were magnetically purified using CS columns (Miltenyi-Biotech, Bergisch Gladbach, Germany) in a SuperMACS II separator (Miltenyi-Biotech, Bergisch Gladbach, Germany) as per manufacturer’s instructions. Columns were blocked with 3% BSA for 15 minutes. One 50mm x 9mm petri dish containing 25 ml of parasites, maintained at 8–10% parasitaemia at a 5% haematocrit produced approximately 6 × 108 infected RBCs at a purity of 80–90%. The parasites were ready to use when in mid/late trophozoites stages. They were washed and resuspended in 20 ml of serum free RPMI and added to the column. Once the parasites had run through the column, it was washed with 25 ml of serum free RPMI. The column was detached from the magnet and the parasites were removed by washing with 50 ml of serum free RPMI.
Parasites were magnetically purified using CS columns (Miltenyi-Biotech, Bergisch Gladbach, Germany) in a SuperMACS II separator (Miltenyi-Biotech, Bergisch Gladbach, Germany) as per manufacturer’s instructions. Columns were blocked with 3% BSA for 15 minutes. One 50mm x 9mm petri dish containing 25 ml of parasites, maintained at 8–10% parasitaemia at a 5% haematocrit produced approximately 6 × 108 infected RBCs at a purity of 80–90%. The parasites were ready to use when in mid/late trophozoites stages. They were washed and resuspended in 20 ml of serum free RPMI and added to the column. Once the parasites had run through the column, it was washed with 25 ml of serum free RPMI. The column was detached from the magnet and the parasites were removed by washing with 50 ml of serum free RPMI.
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