Parasite burden

Parasite burden was measured in the whole spleen of individual mice using a microtitre dilution method adapted from Buffet et al.⁹³ It was necessary to determine parasite burden in the spleen rather than the intestine because it was impossible to harvest immune cells for analysis from the intestine and determine parasite burden in the same animal, however, we have demonstrated previously that the parasite burden in the spleen accurately mirrors that in the intestine⁵⁰. Briefly, prior to the experiment, 96 well plates were seeded with HFF cells and allowed to become confluent. One row was allocated per mouse and each mouse was done in duplicate. Spleens were removed and single-cell suspensions were made by passing through a 70-µm cell strainer. Cells were pelleted at 1500g, and then resuspended in RPMI 1,640 containing 5% FCS at a concentration of 1 × 10⁷ cells/ml. Two hundred microliters of spleen cell suspension was added to the first well of a 96-well plate and then serially diluted 1/2 across the plate. Plates were incubated at 37 °C in 5% CO2 for 7 days before wells were examined for the presence of parasites. A score of parasite burden was allocated based on the last column in which parasites were visible.