Atomic Force Microscopy (AFM) Plasmion-selected trophozoites and schizonts were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde for 30 min on ice and washed twice with PBS. PRBC were immobilized on glass bottom WillCo dishes (WillCo Wells) activated using a low-pressure plasma 02 (Femto, Diener Electronic) [41,42]. The dishes were then covered with poly-L-lysine (Sigma) for 1 hour and rinsed with water. Experiments were conducted at room temperature on a stand alone AFM (Bioscope 2, Veeco Instruments) equipped with a Nonoscope V controller, mounted on an optical inverted microscope (Axiovert 200 M, Zeiss). Bright-field video images were acquired with a CoolSnapES camera (Roper Scientific) driven under MetaMorph software (Molecular Devices). AFM images were acquired in Tapping ModeTM using Veeco’s silicon RTESP Tip (radius of curvature ,10 nm), plane-fitted (1st order), and processed under Vision software to get 3D representation. Height profiles of typical knobs were acquired with Veeco’s integrated software and plotted using R 2.6.1. For each parasite strain three images showing the greatest dispersion in knobs sizes were selected. Adhesion Is Not Linked to clag9 Deletion Statistical Analysis Data were analysed with MinitabH 15 using a 1-way analysis of variance (ANOVA), including Tukey’s test for multiple compar- isons. Results P. falciparum D10 binding assays Previous studies have shown that P. falciparum bulk-cultured parasites can be enriched for their binding to specific endothelial cell receptors [43]. C32 melanoma cells mainly express the CD36 receptor on their surface, and in lesser amounts the receptors ICAM-1 and CSA [44,45]. To determine the adhesive properties of P. falciparum D10 PRBC, we performed binding assays on C32 cells [25]. Asynchronous cultures of P. falciparum D10 and FCR3CD36 were incubated with C32 cells, carefully washed to remove unbound PRBC, and the number of adherent PRBC determined. We observed that D10 PRBC did not bind to the melanoma cells, whereas FCR3CD36-infected erythrocytes were seen to adhere (Fig. 1A and B). Furthermore, no D10 adhesion Figure 1. Binding assay on C32 melanoma cells. A. P. falciparum D10 mature stages are unable to bind to expressed receptors of C32 melanoma cells (CD36&ICAM-1&CSA) compared to the positive control FCR3CD36. B. The graph illustrates the number of bound PRBC per 100 C32 cells for FCR3CD36 (black) and D10 (white). Results are the means 6 standard deviation from five experiments. C. Microscopic images of Giemsa stained FCR3CD36 and D10 on purified receptors. D. Quantification of binding assays for FCR3CD36 (black) and D10 (white) on purified receptors spotted on plastic Petri dishes. Results are the means 6 standard deviation from five experiments. doi:10.1371/journal.pone.0029039.g001 PLoS ONE | www.plosone.org 3 December 2011 | Volume 6 | Issue 12 | e29039