Detailed Invasion Assay Protocol

A detailed protocol for assaying invasion is available in S1 Text. The invasion assay proceeded largely as previously described [59 (link)]. Briefly, parasite lines were treated ±ATc and resuspended in IC buffer, to prevent invasion, and spun down onto the HFF monolayer in an eight-well chamber slide (Ibidi). IC buffer was exchanged for DME/1% FCS/10 mM HEPES, pH 7.5, and incubated for 10 minutes at 37 °C. Samples were then fixed, stained with anti-SAG1 mAb DG52 (1:3,000 dilution), permeabilised, and stained with rabbit anti-GAP45 (1:2,000) [58 (link)], followed by secondary antibodies, then imaging on a Zeiss Live Cell Observer and quantitation by manual counting. For invasion assays using GFP-expressing parasites, GAP45 staining was not carried out. Invasion assays with DCCD were carried out as described above except that parasites were allowed to invade in D1/HEPES medium containing 50 μM DCCD (Sigma). Graphs were generated and statistical tests performed using Prism 7 software.