Invasion of <i>P. falciparum</i> strain 3D7 decreases as untreated RBCs are replaced with enzyme treated RBCs.

<p>RBCs were labeled with either CellTrace DDAO or Violet before enzyme treatment (neuraminidase, trypsin, or chymotrypsin). 1.8×10⁷, 1×10⁷, and 2×10⁶ non-enzymatically treated RBCs (RBC^Ø) were combined with 2×10⁶, 1×10⁷, and 1.8×10⁷ of enzyme treated RBCs (RBC^N, RBC^T, or RBC^C) respectively to achieve 10∶1, 1∶1, and 1∶10 combinations of RBC^Ø to either RBC^N, RBC^T, or RBC^C in the barcoded RBC invasion assay. Invasion assays were inoculated with 2×10⁵ of MACS purified trophozoite stage <i>P. falciparum</i> strain 3D7 parasites and invasion assays were performed as previously described. Data is from a single representative experiment of three independent experiments performed in triplicate. (A) Rate of 3D7 invasion into 1∶10, 1∶1, and 10∶1 combinations of RBC^Ø and either RBC^N, RBC^T, or RBC^C. Bars represent the mean invasion rate and error bars represent the SD. Elongated triangles below the X-axis represent the replacement of RBC^Ø (white triangle) with either RBC^N, RBC^T, or RBC^C (gray triangle) in the total RBC population. Student's <i>t</i>-tests were used to compare invasion rates, *p<0.02 and **p<0.0002. (B, C, and D) Data shows the number of invasion events into RBC^Ø (triangles) and either RBC^N, RBC^T, or RBC^C (circles) as the frequency of each RBC type increases from 10% to 90% of the total RBC population. Linear regression was used to determine the best fit line for <i>P. falciparum</i> invasion of RBC^Ø, RBC^N, RBC^T, and RBC^C. ANCOVA was performed to compare the slopes of the lines fit to <i>P. falciparum</i> invasion of RBC^Ø, RBC^N, RBC^T, and RBC^C. The null hypothesis was no difference between RBC^Ø and either RBC^N, RBC^T, or RBC^C (H₀: β_Ø = β_enzyme, α = 0.05). ANCOVA performed with GraphPad, Prism, v. 5.04, La Jolla, CA calculated a p<0.0001. (E) RBC^Ø datum from panels B–D were superimposed to compare invasion into RBC^Ø when RBC^Ø was combined with RBC^N (circles) RBC^T (triangles) or RBC^C (diamonds). Linear regression was used to determine the best fit line for RBC^Ø invasion data. ANCOVA was performed to compare the slopes of the lines fit to <i>P. falciparum</i> invasion of RBC^Ø. The null hypothesis was that there would be no difference in the invasion of RBC^Ø in the presence of the other RBC populations (H₀: β_Ø = β_enzyme, α = 0.05). ANCOVA performed with GraphPad, Prism, v. 5.04, La Jolla, CA calculated a p<0.2599.