P. Knowlesi erythrocyte invasion assay
Erythrocytes
Blood was collected in 10% citrate phosphate dextrose (CPD) and stored at 4°C unwashed for up to 4 weeks, or washed in RPMI with malaria supplements and stored in malaria culture medium at 50% hematocrit for up to 2 weeks. The DARC+ human erythrocytes used in the erythrocyte binding assay and the P. knowlesi erythrocyte invasion assay had the phenotype Fy(a-b+) as determined by standard blood banking methods using anti-Fya and anti-Fyb antisera (Gamma Biologicals, Houston, TX). Erythrocytes were washed three times in DMEM (Gibco BRL) and resuspended to a hematocrit of 10% in complete DMEM for the erythrocyte binding assay. Erythrocytes used in the P. knowlesi erythrocyte invasion assay were washed three times and resuspended to a hematocrit of 10% using malaria complete RPMI.
Percoll purification of schizont-infected erythrocytes
Cultures of P. knowlesi at 5-10% infected erythrocytes were washed three times in RPMI with malaria supplements and 10% FBS and brought up to a hematocrit of 10%. A 50% Percoll solution was made by adding 0.45 vol 1× PBS, 0.05 vol 10× PBS and 0.5 vol Percoll (Sigma). Two ml of the washed culture was overlaid on 2 ml of the 50% Percoll solution in a 4 ml polystyrene tube and centrifuged for 20 min at 2100 RPM in a Sorvall centrifuge. The ring of cells at the interface was removed, pooled and washed three time in 1× PBS. The pellet was brought up in malaria culture medium to 2 × 107 cells/ml.
P. Knowlesi erythrocyte invasion assay
Human Duffy Fy(a-b+) erythrocytes were washed in complete malaria medium and 2 × 107 washed cells were added to increasing concentrations of sulfated polysaccharide in malaria culture medium at final volume of 900 μl for 1 h at room temperature. To each tube of sulfate polysaccharide-treated erythrocytes, 100 μl or 2 × 106 schizont-infected erythrocytes was added and placed in a well of a polystyrene 24-well plate (Becton-Dickinson). The cultures were maintained under a blood-gas atmosphere at 38°C for 8 h to allow the infected erythrocytes to rupture and release free merozoites capable of infecting new erythrocytes and developing to ring-stage trophozoites. The culture was centrifuged at 2100 RPM for 3 min and a thin smear was made from the pellet. The thin smear was fixed with methanol and stained with Leukostat Solution B (100 mg Eosin Y+ 300 μl 37% formaldehyde +400 mg sodium phosphate dibasic +500 mg potassium phosphate monobasic, q.s. to 100 ml with dH2O), rinsed, and stained with Leukostat Solution C (47 mg Methylene Blue +44 mpp Azure A +400 mg sodium phosphate dibasic +500 mg potassium phosphate monobasic, q.s to 100 ml with dH2O). The percentage of erythrocytes infected with ring-stage trophozoites per 2000 erythrocytes was determined at 1000×. Percentage inhibition of invastion was determined by dividing the percentage of ring-stage parasites at each polyanion concentration by the percentage of ring-stage parasites at 0 μg/ml of the polyanion, multiplying by 100 and subtracting this value from 100.