Macaque, Human, and Reticulocyte-Enriched Blood.

Cynomolgus and rhesus blood was collected by venous puncture into K2 EDTA vacutainers (Becton Dickinson). Human blood (Groups A⁺ and O⁺; Duffy⁺) was obtained from the United Kingdom National Blood Transfusion Service and was used within 2 wk of receipt for all growth invasion assays. Reticulocyte enrichment was achieved using a protocol adapted from a previous method (28, 29), which uses a high K⁺ buffer to prevent reticulocyte dehydration and allows density-based enrichment from normal blood. Human blood cells were washed twice in high K⁺ buffer (1× K⁺ buffer; 20 mM K⋅Hepes, 1 mM MgCl₂, 1 mM NaH₂PO₄, 10 mM glucose, 0.5 mM EGTA, 5 mM NaCl, 116 mM KCl). The washed cells were passed through a sterile CF11 cellulose column (Whatman) under gravity to remove white blood cells, and the column was washed with one volume of K⁺ buffer. Reticulocytes were enriched by centrifugation at 900 × g for 12 min on a cushion of 65% Percoll (GE Healthcare) [stock solution (90% Percoll, 10% 10× K⁺ buffer) diluted into 1× K⁺ buffer]. The RBCs at the layer interface were collected and washed once with K⁺ solution. Ten microliters of RBCs were incubated with 50 µL of reticulocyte stain solution (Sigma) for 15 min; then reticulocytemia was assessed by microscopy on thin blood films, counting reticulocytes (which contain two or more blue-stained granules) and total RBCs.