Cells were counted by inverted optical microscope and
concentration was assessed before being used for IFN-g
ELISpot assay. In all samples, PBMC were >98% viable as
confirmed by Trypan blue (Sigma) exclusion. After isolation,
cell samples not processed immediately were frozen
at 80 8C using a Mr Frosty1 device (Sigma) gradient and
stored in liquid nitrogen for subsequent assays.