CD44 is required for efficient P falciparum invasion. (A) Invasion assays of P falciparum strain 3D7 into WT cRBCs vs CD44-CRISPR cRBCs. Approximately 18 hours after incubation with late-stage schizonts, parasitemia was determined by blinded counting of cytospin slides stained with May-Grünwald and Giemsa stain, and is presented relative to parasitemia in control erythrocytes. Plotted are 4 biological replicates, each of which was performed in triplicate, with the line indicating the mean ± standard error of the mean (SEM); ∗P = .0062, 2-tailed paired t test. (B) Cytospin images of P falciparum parasites ∼18 hours after invasion into isogenic WT or CD44-CRISPR cRBCs. (C) Flow cytometry plot showing efficiency of negative selection for CD44-null cRBCs using CD44 microbeads. Anti-CD44 antibody BRIC 222 (IBGRL) was used at 1:10 000 followed by goat anti-mouse IgG-488 (1:1500). (D) Invasion assays of P falciparum strain 3D7 into WT cRBCs vs CD44-null cRBCs isolated by negative selection. Parasitemia was determined by blinded counting of cytospin slides by 2 individuals, and is presented normalized to the parasitemia in the WT cRBCs for each replicate. Each of 4 biological replicates are plotted, each of which were performed in triplicate, with the line indicating the mean of the biological replicates, ±SEM; ∗∗P = .0010, 2-tailed t test.