General adhesion assay

Static protein assays were carried out as described by McCormick ((McCormick et al. 1997). Briefly, 2 µl of purified recombinant protein chimeras of wild-type ICAM- 1 (ICAM-1Ref_{) and CD36 proteins were spotted onto 60 mm diameter} bacteriological Petri dishes and incubated in a humidified chamber for 2 hr at 37o_C. Proteins were aspirated off, and dishes were blocked overnight at 4o_{C in 1%} BSA/PBS. Blocking solution was removed, the dish washed in binding buffer and 2 ml of parasite suspension at 3% parasitemia and 1% hematocrit added to each dish. Dishes were incubated at 37o_{C for 1 hr, with re-suspension for every 10 min.} Unbound pRBC and RBC were removed by repeated washing, bound cells fixed with 1% glutaraldehyde for 1 hr and then stained with 5% giemsa for 20 min. Level of adhesion were viewed under 10 x 100 magnification fields microscopically, and photo was taken and was analyzed using Image-Pro Plus image analyzer software version 5.1. From this the number of adherent pRBC per mm2 _{was calculated.}

Flow protein adhesion assay (microslide)

Flow reversal assays were carried out on microslides (3- aminopropyltriethoxysilane (APES)-treated) coated with ICAM-1Ref _{at 50 µg/ml} (Berendt et al. 1992). Microslides were pre-prepared by coating with APES following nitric acid treatment to allow for protein/cell adherence, then autoclaved. Before the experiment, a small piece (1.5 cm) of thin wall rubber tubing was attached to one end of the microslide, marked to indicate orientation of the coated surface, and slides were autoclaved. Slides were then coated with ICAM- 1Ref _{at 50 μg/ml by drawing up 65 μl of solution using a pipette tip connected to} the tubing. Protein was allowed to adhere to the microslide by incubation in a humid 37°C chamber for 2 hours then washed by drawing through 4 x 65 μl of PBS with 1% BSA Slides were blocked in 1 % BSA overnight at 4°C. Parasites were prepared as for static protein assay and everything warmed to 37°C before use. The flow system was based on that described by Nash et al (Nash et al. 1992). A

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suspension of pRBC at 3% parasitemia and 1% hematocrit were flowed through the microslide for 5 min to allow for pRBC adhesion. Flow was continuous throughout the experiment at 0.05 Pa (0.186 ml/min) shear stress. After 5 min PRBC flow, flow was switched to binding buffer alone to remove unbound pRBC for another 2 minutes. The number of adherent pRBC was counted in six separate fields under 20 x 100 magnification fields. From this the number of adherent pRBC per mm2 was calculated. To enable re-use of the slide parasites were removed by

washing the slide through 2 x 2 ml with water then 2 x 2 ml binding buffer. To ensure this had no effect on binding, a positive control was carried out as the last run on a slide in each experiment. Slides were not re-used more than 6 times.

Static cell cdhesion assay

Static cell assays were carried out as described by Gray et al 2003. Endothelial cells, HUVEC and HDMEC (2-6th _{passage) at 5 x 10}-3 _{cells were seeded onto 1%} gelatin coated 13 mm Thermanox coverslips (Nalgene, Nunc). After leaving overnight for cells to adhere the medium was changed and cells cultured until confluent (2 – 5 days). Once confluent, the cells were incubated overnight at 37o_C with or without 1 ng/ml TNF (Biosource International). Cells were washed with binding buffer and incubated with 0.5 ml of parasite suspension (3% parasitemia, 1% HCT) for 1 h at 37o_{C, with gentle resuspension every 10 min. A one hour} gravity wash was used to remove unbound cells. Adherent cells were fixed using 1% glutaraldehyde for 1 hr and then stained with 5% giemsa for 20 min. Coverslips were dried and mounted on slides using DPX mountant. Level of adhesion was quantitated microscopically under 20 x 100 magnification fields. From this the number of adherent pRBC per mm2 _{was calculated.}

Flow cell adhesion assay (chamber slide)

Binding assays under flow conditions were carried out as described by Gray et al 2003. This type of assay attempts to mimic the conditions seen in the post capillary venule by allowing infected RBC to flow over chamber slides coated with endothelial cells with constant flow rate at 0.24 ml/min (0.05Pa). The slides were

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coated with 1% gelatin for 1 hr at 37o_{C and further seeded with 5 x 10}-3 _{cells and} were kept in a CO2 incubator overnight. One confluent T25 flask of endothelial cells was trypsinised and used to seed 3 (for next day assay) or 6 (For 2-4 days growth before assay) chamber flasks. 2.5 ml trypsinised cells were added per flask. After settling overnight the medium was changed and cells cultured until confluent. Confluent chamber slides were then incubated overnight with or without 1 ng/ml TNF prior to use. Parasite suspensions (3% parasitemia and 1% HCT) were flowed over for a total 5 min, and flowed by binding buffer for another 2 min to remove the unbound pRBC. The flow rate yielded a wall shear stress of 0.05 Pa (o.24 ml/min), which has been used widely to mimic wall shear stresses in the microvasculature. The number of adherent pRBC was counted in six separate fields under 20 x 100 magnification fields. From this the number of adherent pRBC per mm2 _{was calculated.}