Elisas specific for ifnγ

To measure antigen-specific cytokine production, retroauricular LNs were isolated and single cell suspensions prepared. One million LN cells in 200 µl complete RPMI 1640 medium (BioWhittaker) were cultured in the presence of 25 µg/ml SLA. Supernatants were harvested 48 h after stimulation and analyzed with ELISAs specific for IFNγ (R&D Systems), IL-4 and IL-10 (BD). IFNγ and IL-4 levels in serum samples were measured the same way.

C57BL/6, µMT, IgGi, and IgMi mice were infected with physiological low dose of 10³ metacyclic L. major promastigotes in a volume of 10 µL by intradermal injection into both ears using 0.3-mm-diameter needles. In some experiments, parasites were opsonized for 10 min with either NMS, IS, or α-PL and washed. In others, mice were reconstituted i.p. two times with 100 µL of either NMS or IS.
Lesion volumes were measured weekly in three dimensions and are reported as ellipsoids [(a/2 × b/2 × c/2) × 4/3 × π]. Parasites present in lesional tissue were enumerated using a limiting dilution assay as previously described [11 (link)].
For measurement of antigen-specific cytokine production, draining LN cells of infected C57BL/6 mice were recovered and single-cell suspensions were prepared. One-million LN cells/200 μL complete RPMI 1640 (Biochrome) were added to 96-well plates in the presence of 25 μg/mL SLA. Supernatants were harvested 48h after stimulation and assayed using ELISAs specific for IFNΓ (R&D Systems), as well as IL-4 and IL-10 (BD).